ABSTRACT The response of childhood acute lymphoblastic leukemia (ALL) to dexamethasone predicts the long-term remission outcome. To explore the mechanisms of dexamethasone resistance in B

cell ALL (B-ALL), we generated dexamethasone-resistant clones by prolonged treatment with dexamethasone. Using RNA-sequencing and high-throughput screening, we found that

dexamethasone-resistant cells are dependent on receptor tyrosine kinases. Further analysis with phosphokinase arrays showed that the type III receptor tyrosine kinase FLT3 is constitutively

active in resistant cells. Targeted next-generation and Sanger sequencing identified an internal tandem duplication mutation and a point mutation (R845G) in FLT3 in dexamethasone-resistant

FLT3 might be useful for the treatment of relapsed B-ALL patients. SIMILAR CONTENT BEING VIEWED BY OTHERS GLUCOCORTICOIDS PARADOXICALLY PROMOTE STEROID RESISTANCE IN B CELL ACUTE

LYMPHOBLASTIC LEUKEMIA THROUGH CXCR4/PLC SIGNALING Article Open access 29 May 2024 DASATINIB OVERCOMES GLUCOCORTICOID RESISTANCE IN B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA Article Open access 22

May 2023 MOLECULAR AND PHARMACOLOGICAL HETEROGENEITY OF _ETV6_::_RUNX1_ ACUTE LYMPHOBLASTIC LEUKEMIA Article Open access 29 January 2025 INTRODUCTION Acute lymphoblastic leukemia (ALL) is

one of the most common childhood cancers and can originate both from the B-lineage (B-ALL) and the T-lineage (T-ALL). Glucocorticoids, such as dexamethasone and prednisolone, are important

drugs for the treatment of ALL.1 In combination with chemotherapeutic agents, glucocorticoids help to achieve clinical remission, and sensitivity to glucocorticoids is considered as a

positive prognostic indicator. Patients unresponsive to glucocorticoids often relapse and display poor prognosis. Therefore, understanding the mechanisms behind glucocorticoid insensitivity

is important and will help us to develop novel therapeutic modalities. In ALL glucocorticoids induce apoptosis, which is mediated through binding to the glucocorticoid receptor (GR). GR is a

nuclear receptor that also acts as a transcription factor. Upon glucocorticoid binding, GR inhibits activator protein-1 (AP-1)- and nuclear factor-κB (NF-κB)-regulated gene transcription,

and at the same time promotes glucocorticoid-responsive element-driven gene transcription.2 Besides, inhibition of AP-1- and NF-κB-regulated gene transcription, cooperation between AP-1 and

GR in transcription,3 and crosstalk between NF-κB and GR4,5 have been reported, which suggests a context-dependent regulation of AP-1 and NF-κB rather than only inhibitory effects.

Glucocorticoids are useful drugs to induce apoptosis in ALL and have also been widely used to treat inflammatory disorders. However, prolonged use leads to the emergence of glucocorticoid

resistance.6 The mechanisms of glucocorticoid resistance in leukemia have been studied extensively. Both regulation of expression and function of GR can contribute to glucocorticoid

resistance. For instance, activation of NOTCH1 signaling inhibits auto-upregulation of GR expression. Therefore, pharmacological inhibition of NOTCH1 restores glucocorticoid sensitivity.7

The relapse-associated mutation in _NR3C1_ results in the expression of a non-functional receptor and thereby impairs glucocorticoid sensitivity.8 Furthermore, aberrant activation of the

PI3K/mTOR pathway has been linked to glucocorticoid resistance in T-ALL.9 This is partially mediated by AKT, which phosphorylates GR on S134 and thereby blocks nuclear localization of GR.10

Mutations in the transcriptional coactivator CREBBP transcriptionally regulates glucocorticoid-responsive genes, suggesting that functional CREBBP is required for glucocorticoid

sensitivity.11 Inhibition of glutathione synthesis restored dexamethasone sensitivity in the dexamethasone-resistant B-ALL cell line 697,12 suggesting the existence of additional mechanisms

of dexamethasone resistance. In this report, we show that cells resistant to dexamethasone harbor activating mutations in the receptor tyrosine kinase FLT3. RESULTS PROLONGED DEXAMETHASONE

TREATMENT INDUCES DEXAMETHASONE RESISTANCE IN B-ALL CELLS In order to understand how long-term dexamethasone treatment affects B-ALL cells, we used three dexamethasone-sensitive cell lines:

697 (half-maximal effective concentration (EC50) = 8.2 nM), NALM-6 (EC50 = 3.9 nM), and RS4;11 (EC50 = 1.5 nM), and the dexamethasone-insensitive cell line TANOUE (EC50 >10 µM). These

cell lines were cultured with an increasing concentration of dexamethasone for 90 days. In parallel, another set of cell lines was cultured with an equivalent amount of dimethyl sulfoxide

(DMSO) (which was used to dilute dexamethasone). After 90 days, cells were cultured in normal growth medium for 2 weeks and EC50 was measured. We observed that all three

dexamethasone-sensitive cell lines cultured in the presence of dexamethasone became highly resistant to dexamethasone, while DMSO-treated cells were still sensitive (Fig. 1a). The relation

between dexamethasone sensitivity and GR expression does not always correlate.13,14 Therefore, we first checked the GR expression in both dexamethasone-sensitive and -resistant cell lines.

The expression of GR remained unchanged in TANOUE cells, while it was reduced in 697 and NALM-6 cells (Fig. 1b). However, while the most sensitive cell line, RS4;11, showed strong GR

expression, its expression was completely lost in the corresponding resistant cells (Fig. 1b). These data are in line with previous reports that GR expression is one of the factors relating

to dexamethasone sensitivity but not the only factor.13,14 DEXAMETHASONE-RESISTANT RS4;11 CELLS ARE SENSITIVE TO RTK INHIBITORS To understand the molecular differences between

dexamethasone-sensitive and -resistant cell lines, we used RNA-sequencing (RNAseq). We observed that the gene expression patterns were mostly identical between sensitive and resistant lines

of 697, NALM-6, and TANOUE cells, whereas RS4;11 cells showed a more scattered expression pattern indicative of differences in gene expression between the two types of cells (Fig. 2a). These

data suggest that both dexamethasone-sensitive and -resistant lines of 697, NALM-6, and TANOUE cells keep similar gene expression pattern, while RS4;11-resistant cells show a major

difference compared to its parental cell line. As we observed a major variation in gene expression of RS4;11, we checked the pathway enrichment in resistant cells using RNAseq data. We

observed enrichment of several kinase and cytokine signaling pathways in resistant RS4;11 cell line (Fig. 2b). Since we observed enrichment of kinase and cytokine signaling pathways in the

dexamethasone-resistant RS4;11 cell line, we hypothesized that there is a switch in the dependency of RS4;11 cells from dexamethasone to kinase-related signaling. To identify the possible

kinase dependency of RS4;11 cells, we used a panel of 378 inhibitors against different kinases. Both sensitive and resistant lines of NALM-6, 697, and TANOUE cells displayed similar response

to the inhibitors, but the resistant RS;411 cell line displayed increased sensitivity to several receptor tyrosine kinase (RTK) inhibitors compared to the corresponding sensitive cell line

(Fig. 2c). Taken together, these data suggest that the mechanism behind the resistant phenotype of RS4;11 is different from that of NALM-6, 697, and TANOUE cell lines.

DEXAMETHASONE-RESISTANT RS4;11 CELLS DISPLAY TYROSINE PHOSPHORYLATION OF FLT3 Since we did not observe any major differences between the gene expression and kinase inhibitor response, we

suggest that the resistance of 697 and NALM-6 is probably mediated by reduced expression of GR or due to a loss-of-function mutation in GR. Several other mechanisms have also been described

and discussed in the Introduction section.7,8,9,10,11,12 However, the difference in gene expression in the two RS4;11 cell lines and their differential response to kinase inhibitors evoked

our interest. Coinciding with the development of a resistant phenotype, the RS4;11 cells completely lost GR expression. Most likely this is due to the fact that a small fraction of cells

that initially were lacking GR expression were selected for during the long-term exposure to dexamethasone, and that selected for cells that carry different genetic mutations. Since we

observed that dexamethasone-resistant RS4;11 cells are sensitive to several RTK inhibitors, we checked for activation of RTKs in this cell line using a human proteome phospho-RTK array.

Surprisingly, we observed strong tyrosine phosphorylation of FLT3 and weak tyrosine phosphorylation of AXL in resistant cells, which could not be seen in sensitive cells (Fig. 3a).

Furthermore, using a phosphokinase array we observed that phosphorylation of ERK1/2 and of CREB at S133 was enhanced in resistant cells (Fig. 3b). Collectively, our data suggest that

dexamethasone-resistant RS4;11 cells display dependency of constitutively active RTK signaling. DEXAMETHASONE-RESISTANT RS4;11 CELLS CARRY ONCOGENIC MUTANTS OF FLT3 AND RESPOND TO FLT3

INHIBITION We then checked the expression of FLT3 and AXL in RS4;11 cell lines. We observed strong expression of FLT3 in dexamethasone-sensitive RS4;11, where the fully glycosylated, mature

FLT3 band was stronger than the partially glycosylated, immature band (Fig. 4a), which is a characteristic of cells expressing wild-type FLT3. The observation that the partially

glycosylated, immature FLT3 band was stronger in dexamethasone-resistant RS4;11 cells (Fig. 4a) raised the possibility that the resistant cells carry an oncogenic internal tandem duplication

(ITD) mutation in FLT3, which typically gives this pattern of expression.15,16 This is also supported by the fact that resistant RS4;11 cells showed constitutive tyrosine phosphorylation of

FLT3 as well as constitutive STAT5 phosphorylation (Fig. 4b) and that the second-generation FLT3 inhibitor AC220 could block tyrosine phosphorylation of both FLT3 and STAT5 (Fig. 4c).

DEXAMETHASONE-RESISTANT RS4;11 CELLS CARRY FLT3-ITD AND FLT3-R845G MUTATIONS To verify the presence of FLT3 mutations and also in order to see whether any other oncogenic mutations exist in

RS4;11 cells, we used targeted sequencing of 600 cancer-related genes. We identified a FLT3 point mutation (c.2533A>G, R845G, ratio 65%, coverage 1504×) and an FLT3-ITD mutation

(p.E598_Y599insFDFREYE 22%, coverage 487×) (Fig. 5a). The point mutation was further confirmed by Sanger sequencing (Fig. 5b). FLT3-ITD is a well-studied oncogenic mutation and R845G has

also been shown to be a constitutively activating mutation.17 DEXAMETHASONE-RESISTANT RS4;11 CELLS ARE SENSITIVE TO THE SECOND-GENERATION FLT3 INHIBITORS AC220 AND CRENOLANIB Since

dexamethasone-resistant RS4;11 cells harbor oncogenic mutations in FLT3, we have tested the possibility of using the second-generation FLT3 inhibitors AC220 and crenolanib. Both the

inhibitors significantly reduced the growth of dexamethasone-resistant RS4;11 cells, while the growth of dexamethasone-sensitive RS4;11 and 697 cells or dexamethasone-resistant 697 cells

remained unchanged (Fig. 6a, b). Furthermore, in a mouse xenograft model, crenolanib delayed tumor formation of dexamethasone-resistant RS4;11 cells (Fig. 6c, d). Taken together, data

suggest that dexamethasone-resistant RS4;11 cells are dependent on the activity of oncogenic FLT3 signaling. DISCUSSION In this study, we used three dexamethasone-sensitive B-ALL cell lines

from three different genetic backgrounds to generate dexamethasone-resistant cell lines. Although the 697 cell line carries an _E2A-PBX1_ (_TCF3-PBX1_) fusion, RS4;11 carry an _MLL-AF4_

fusion and NALM-6 carry an _NRAS_ mutation, all three cell lines displayed similar sensitivity to dexamethasone (EC50 <10 nM). E2A-PBX1 fusion acts as a constitutively active

transcription factor that downregulates the expression of _CDKN2A_.18 _CDKN2A_ encodes two distinct proteins (p16INK4A and p14ARF), which are well-known regulators of the cell cycle.

E2A-PBX1 does not act as a transcriptional repressor, but this fusion protein enhances expression of _BMI-1_,18 which is known to be a lymphoid oncogene and functions as a transcriptional

repressor. On the other hand, MLL-AF4 suppresses the expression of another cell cycle regulatory protein, p27KIP1 through direct transcriptional repression of _CDKN1B_.19 Furthermore, ALL

patients with _MLL-AF4_ rearrangements overexpress _HOXA9_,20 which is a transcription factor that has been shown to be important for the proliferation and survival of _MLL_-rearranged

leukemias.21 HOXA9 mediates upregulation _BCL2_ expression, which in turn provides survival signals to the leukemic cells.22 Therefore, combinatorial use of dexamethasone and BCL2 inhibitor

displayed a synergistic effect in inhibition of leukemia.23 Although patients with _MLL-AF4_ fusion respond to glucocorticoid-based chemotherapy, this group of patients are considered to

have a poor prognosis and have about 60% disease-free survival.24 Current studies suggest that this group of patients shows resistance to glucocorticoids, which has been shown to be at least

partially mediated by constitutive activation of mitogen-activated protein kinase signaling.25,26,27 Here we provide evidence that cells that are resistant to dexamethasone display

constitutive activation of RTK signaling. All three dexamethasone-sensitive B-ALL cell lines became resistant during 90 days treatment of dexamethasone, suggesting that prolonged

treatment-induced resistance to dexamethasone in vitro. This was independent of the GR expression levels as dexamethasone-resistant 697 cells also express a similar level of GR as sensitive

cells. However, while both 697 and NALM-6 cells kept a certain level of GR expression after 90 days of dexamethasone treatment, expression was almost lost in RS4;11 cells. Besides that,

RS4;11 cells displayed a major deviation with respect to gene expression and kinase inhibitor sensitivity when comparing the dexamethasone-sensitive and -resistant cells, suggesting that

this cell line harbors a different mechanism of dexamethasone resistance than the other two cell lines. Furthermore, RS4;11 cells resistant to dexamethasone showed constitutive activation of

FLT3. A relationship between _MLL_ rearrangement and FLT3 expression has been established in several studies. For example, FLT3 expression was found to be consistently higher in ALL

patients positive for _MLL_ rearrangement,28 and mutations in the activation loop of FLT3 that confer constitutive activation of FLT3 was identified in 17% of _MLL_-rearranged ALL

patients.29 However, higher FLT3 expression and oncogenic mutations are not exclusive to _MLL_-rearranged ALL, while it occurs frequently in hyperdiploid ALL and less frequently in

_TEL-AML1_ fusion ALL.30,31 We observed that the 697 and RS4;11 cell lines express higher levels of FLT3, while its expression was undetectable in TANOUE cells and low in NALM-6 cells. Since

dexamethasone-sensitive RS4;11 cells do not have any FLT3 activation, and since it is unlikely that dexamethasone will induce mutation in _FLT3_, it seems that a small fraction of RS4;11

cells carry FLT3 mutations from the beginning. Dexamethasone selection probably selects for cells that are dependent on FLT3 signaling. Collectively, our data suggest that

dexamethasone-resistant RS4;11 cells are a subpopulation of B-ALL cells that carry FLT3-ITD or R845G mutations, and therefore it could prove useful to screen B-ALL patients who are resistant

to dexamethasone for mutations in FLT3, which then could be targeted with FLT3 inhibitors that are already available on the market. METHODS ANTIBODIES AND CHEMICALS Anti-GR (sc-8992; 1:1000

dilution), anti-β-actin (sc-47778; 1:1000 dilution), anti-AXL (sc-1096; 1:1000 dilution), anti-FLT3 (sc-479; 1:1000 dilution), and anti-STAT5 (sc-835; 1:1000 dilution) were from Santa Cruz

Biotechnology (Dallas, TX, USA). Anti-phospho-tyrosine antibody 4G10 (05-321; 1:1000 dilution) was from Millipore. Dexamethasone (D4902, Sigma-Aldrich, St. Louis, MI, USA) was dissolved in

DMSO. All uncropped blots are available in supplementary figure. CELL CULTURE AND GENERATION OF DEXAMETHASONE-RESISTANT B-ALL CELL LINES The B-ALL cell lines RS4;11, 697, NALM-6, and TANOUE

were purchased from the DSMZ (Braunschweig, Germany). B-ALL cell lines were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100U/ml penicillin

and 100 µg/ml streptomycin. All B-ALL cell lines were treated with dexamethasone and doses were doubled when the treated cells started to proliferate at an equal rate to the untreated

parental cells. The doses were increased at regular intervals until 5 µM concentration was reached. The resistant cells were further grown for 2 weeks in the absence of inhibitors. DRUG

SENSITIVITY ASSAY Sensitive and resistant B-ALL cells were grown in RPMI-1640 medium supplemented with 10% FBS, 100U/ml penicillin, and 100 μg/ml streptomycin. Cells were then seeded in

96-well plates (10,000 cells per well) in the presence of different concentrations of dexamethasone. After 48 h, 10 μl of PrestoBlue was added to each well, followed by 2 h of incubation.

Cell viability was calculated according to the manufacturer’s protocol. A kinase inhibitor library including 378 kinase inhibitors was obtained from Selleck Chemicals (Houston, TX, USA).

Stock solutions of 10 mM inhibitor were diluted to 100 nM using the cell culture medium. Cell viability assays using PrestoBlue were used to examine the effect of inhibitors. PHOSPHOKINASE

ARRAYS Proteome Profiler Human Phospho-RTK Array Kit (ARY001B) and Proteome Profiler Human Phospho-Kinase Array Kit (ARY003B) were obtained from R&D System (Minneapolis, MN, USA).

Dexamethasone-sensitive and -resistant cells were lysed and the lysates were processed according to the manufacturer’s protocols. TARGETED SEQUENCING OF CANCER PANEL INVIEW Oncopanel

All-in-one service from Eurofins Genomics provided analysis of 597 key cancer-specific genes. Total genomic DNA from dexamethasone-sensitive and -resistant cells was purified using Qiagen

DNeasy Blood and Tissue Kit (69504), and then sent to Eurofins Genomics for processing. MOUSE XENOGRAFT STUDIES Ten female non-obese diabetic/severe combined immunodeficiency gamma (NSG)

mice (each weighing ~20 g and housed by the Laboratory Animal Facilities at Medicon Village, Lund University) were injected with 2,000,000 cells with 1:1 Matrigel subcutaneously. Mice were

then divided into two groups and treated either with crenolanib or with the vehicle. One week after injection of cells, mice were treated alternative days by intravenous injection of 12 

mg/kg crenolanib or vehicle for additional days until the tumor reached a size of 1 cm3 (8th to 16th day). Drug efficacy was checked by monitoring the tumor growth in both groups and by

regularly measuring the body weight and tumor volume of the mice. Mice were sacrificed after the size of the tumors had reached about 1 cm3. ETHICAL CONSIDERATION Mice were maintained by

following regulation approved by the Lund University. All animal experiments were performed under an ethical permit from the Swedish Animal Welfare Authority. RNASEQ ANALYSIS The RNA quality

was analyzed using a Bioanalyzer (Agilent) and samples with an RNA integrity greater than seven were further analyzed. Subsequently, RNAseq was performed using TruSeq Stranded mRNA Kit for

NeoPrep from Illumina and the sequencing was performed using Illumina NextSeq 500 instrument. RNAseq data analysis was performed using a pipeline where Demultiplexing step involves in

reorganizing the FASTQ files based on the sample index information, and generating the statistics and reporting files, which was performed using the bcl2fastq2 software (Illumina), Masking

step involves filtering ribosomal RNA (GenBank loci NR_023363.1, NR_003285.2, NR_003286.2, NR_003287.2, X12811.1, U13369.1), PhiX (phiX174), and Illumina control (NC_001422.1), and repeat

sequence analysis was performed using the bowtie2 program. Mapping steps, after filtration of remaining reads, were aligned to the human genome reference (UCSC hg38 build), performed using

TopHat2 program. Expression count step, expression levels, and fragments per kilobase of exon per million mapped reads were calculated using Cufflinks 2.2.1 program. STATISTICAL ANALYSIS

Statistical analysis was performed using GraphPad Prism 5.0. One-way analysis of variance or Student’s _t_ test was used where needed. REPORTING SUMMARY Further information on research

design is available in the Nature Research Reporting Summary. DATA AVAILABILITY All raw data are available upon request. RNAseq data are available at the ArrayExpress (E-MTAB-7781). Raw data

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leukemia. _Blood_ 103, 3544–3546 (2004). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We would like to thank Professor Lars Rönnstrand for comments on the manuscript.

This research was the Kungliga Fysiografiska Sällskapet i Lund (S.A.M. and K.S.), Ollie and Elof Ericsson’s Stiftelse (J.U.K.), the Crafoord Foundation (J.U.K.), Magnus Bergvalls Stiftelse

(J.U.K.) and the Swedish Childhood Cancer Foundation (J.U.K.). J.U.K. is a recipient of an Assistant Professorship (forskarassistenttjänst) grant from the Swedish Childhood Cancer

Foundation. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Division of Translational Cancer Research, Department of Laboratory Medicine, Lund University, Lund, Sweden Rohit A. Chougule, 

Kinjal Shah, Sausan A. Moharram & Julhash U. Kazi * Division of Oncology and Pathology, Department of Clinical Sciences Lund, Lund University, Lund, Sweden Johan Vallon-Christersson

Authors * Rohit A. Chougule View author publications You can also search for this author inPubMed Google Scholar * Kinjal Shah View author publications You can also search for this author

inPubMed Google Scholar * Sausan A. Moharram View author publications You can also search for this author inPubMed Google Scholar * Johan Vallon-Christersson View author publications You can

also search for this author inPubMed Google Scholar * Julhash U. Kazi View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS R.A.C., K.S.,

S.A.M., and J.V.-C. performed the experiments. J.U.K. supervised the research. R.A.C., K.S., S.A.M., J.V.-C., and J.U.K. analyzed the data. J.U.K., R.A.C., S.A.M., J.V.-C., and K.S. wrote

the manuscript. All authors read and approved the final manuscript. The authors declare that they have no conflict of interests. CORRESPONDING AUTHOR Correspondence to Julhash U. Kazi.

ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE: Springer Nature remains neutral with regard to jurisdictional

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Glucocorticoid-resistant B cell acute lymphoblastic leukemia displays receptor tyrosine kinase activation. _npj Genom. Med._ 4, 7 (2019). https://doi.org/10.1038/s41525-019-0082-y Download

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