A novel subclonal rearrangement of the STRN3::PDGFRB gene in de novo acute myeloid leukemia with NPM1 mutation and its leukemogenic effects

Chromosome translocations in the 5q31-33 region are associated with a range of hematologic malignancies, some of which involve the platelet-derived growth factor receptor beta (PDGFRB) gene. We report a case of acute myeloid leukemia (AML) with a mutation in the NPM1 gene (NPM1-mut AML) and a subclonal gene rearrangement involving the PDGFRB gene. We identified a novel fusion gene, STRN3::PDGFRB, resulting from t(5;14) (q32;q12) chromosomal rearrangement. Sequential FISH confirmed that ~15% of leukemic cells carried the PDGFRB gene rearrangement, which suggests that STRN3::PDGFRB is a previously unreported fusion gene in a subclone. Reverse transcription PCR (RT-PCR) and Sanger sequencing confirmed that the fusion gene consisted of STRN3 exon 7 fused to PDGFRB exon 11, resulting in a chimeric protein containing the coiled-coil domain of striatin-3 and the transmembrane and intracellular tyrosine kinase domains of the PDGFRB. The new protein exhibited distinct cytoplasmic localization and had leukemogenic effects, as demonstrated by its ability to transform Ba/F3 cells to growth factor independence and cause a fatal myelodysplastic/myeloproliferative neoplasm (MDS/MPN)-like disease in mice, which then transformant to T-cell lymphoblastic lymphoma in secondary recipients. Ba/F3 cells expressing STRN3::PDGFRB or ETV6::PDGFRB were sensitive to tyrosine kinase inhibitors (TKIs) and selinexor, but in vitro experiments showed that the combination of imatinib and selinexor had a marked synergistic effect, although only the imatinib alone group could prolong the survival of T-cell blast transformation recipient mice. Our findings demonstrate the leukemogenic effects of the novel fusion gene and provide insights into the clone evolution of AML, which can be influenced by therapy selection. Furthermore, our results provide insight into the potential therapeutic options for patients with this type of mutation, as well as the need for careful consideration of treatment selection to prevent undesirable side effects.


INTRODUCTION
According to the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer, over 50 variants of PDGFRB fusion partners have been identified (Supplementary Table 1) [1].Hematologic malignancies with PDGFRB rearrangement are usually classified as "Myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions (MLN-TK)" in the 5th World Health Organization (WHO) classification of hematopoietic neoplasms [2] and International Consensus Classification (ICC) [3].However, some patients with acute lymphocytic leukemia (ALL) with PDGFRB rearrangements are classified as Ph-like ALL [4].The fusion oncoproteins induced by PDGFRB rearrangement exhibit characteristics of having a C-terminal tyrosine kinase constitutively activated by an N-terminal dimerization domain from the fusion partner protein.The STRN3 gene at 14q12 has been identified as a recurrent fusion partner of JAK2 in Ph-like ALL [5].Striatin-3, encoded by STRN3, is composed of a coiled-coil domain, a calmodulin-binding site, a caveolin-binding motif, and a WDrepeat domain, and can associate with additional proteins to form a large protein complex [6].For example, it forms a PP2A-STRN3 phosphatase complex with protein phosphatase 2A (PP2A), which inhibits the Hippo tumor suppressor by affecting the dephosphorylation of MST1/2, and participates in the occurrence and development of gastric cancer and other tumors [6].NPM1-mut AML is a distinct genetic entity in the 5th WHO and ICC classification [2].NPM1 mutation frequently co-occur with mutations of FLT3, DNMT3A, and IDH1/2 genes [7,8], and the prognosis and treatment of NPM1-mut AML depend on the cooccur mutations [9].Most NPM1-mut AML cases present a normal karyotype.Chromosomal abnormalities, in approximately 14% of cases of NPM1-mut AML, are probably secondary, as indicated by the frequent occurrence of cells with an abnormal karyotype as subclones within the population with a normal karyotype, particularly +8, del(9q), +4 [10].
AML arises in a founding clone that occasionally gives rise to subclones, but the subclone with tyrosine kinase gene rearrangement (TKR) is rare.Single cases of AML were reported to carry a BCR::ABL1 subclone, in coincidence with CBFB::MYH11 [11], RUNX1::RUNX1T1 [12] or NPM1-mut [9,12,13].Here, we identified the STRN3 gene as a novel PDGFRB partner in the subclone of an NPM1-mut AML patient.The fusion gene is sufficient to cause an MDS/MPN-like disease rather than AML in retroviral murine models, which is characterized by hepatosplenomegaly, extramedullary hematopoiesis, and neutrophilic leukocytosis.The T-cell transformation occurred in our model upon serial passage, and the imatinib effectively controlled the disease.For AML patients with specific subclones (e.g., tyrosine kinase gene rearrangement, FLT3-ITD), the treatment is fraught with controversy, and the prognostic impact of these subclones remains unclear.There are certain diagnostic and therapeutic problems, which are summarized and discussed here.

MATERIALS AND METHODS Patient specimen preparation
The patient samples were obtained from the bone marrow or hair follicles at the time of diagnosis, remission, and relapse.Patient consent was provided and studies using patient samples were approved by the ethical advisory board of the Institute of Hematology and Blood Diseases Hospital.This study was conducted in accordance with the Declaration of Helsinki.

Cytogenetic and molecular cytogenetic studies
Chromosome analysis was performed on bone marrow (BM) cells using standard cytogenetic techniques and G-banding.A total of 20 metaphase cells were analyzed.Karyotypes were prepared using Applied Imaging CytoVision software and described according to the guidelines of the International System for Human Cytogenetic Nomenclature (ISCN, 2016) [14].
FISH studies were performed on fixed cultured cells, using a standard AML probe panel with 200 cells scored for each probe set.After the identification of the t(5;14), additional FISH testing was carried out by performing sequential hybridization with a PDGFRB dual-color break-apart probe (Vysis, IL, USA).

Next-generation sequencing (NGS)
RNA-Seq libraries were generated with the NEB Next® UltraTM RNA Library Prep Kit (NEB, USA).Whole genome sequencing (WGS) libraries were prepared using NEB Next® UltraTM DNA Library Prep Kit (NEB, USA).Exon capture was performed using the IDT xGen Exome capture kit (Integrated DNA Technologies, Coralville, IA, USA) and DNA libraries were prepared following the manufacturer's instructions.The targeted gene panel was constructed using a custom IDT design (Integrated DNA Technologies, Coralville, IA, USA), covering hematologic neoplasmsrelated exonic regions of 150 genes (Supplementary Table 2).All libraries were sequenced using an Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA).Raw reads of RNA-seq were aligned to the human reference genome (GRCh37/hg19) using Hisat2 (v2.0.5), followed by SATR-fusion software (1.9.0) to detect fusion genes.Raw reads of WGS and target capture sequencing (TES) were aligned to the human reference genome (GRCh37/hg19) using Burrows-Wheeler Aligner (BWA) software [15].The somatic SNV was detected by muTect [16], the somatic InDel by Strelka [17], and the somatic SV by Manta [18].Control-FREEC was used to detect somatic CNV [19].The allelic ratio (AR) of FLT3-ITD to wild-type alleles was further examined using PCRcapillary electrophoresis, and a defined mutant/wild-type >0.50 was considered as high AR according to previous studies [20].We used the R package sciClone to estimate mutational clusters and Infer clonal architecture [21].The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive in BIG Data Center, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China, under the accession number HRA001233 and HRA004121.

Reverse transcription-polymerase chain reaction (RT-PCR) of fusion breakpoints and Plasmid constructs
Total RNA was isolated from the BM mononuclear cells (BMMNCs) using RNAiso Plus (Takara, Dalian, China) according to the manufacturer's protocol.After quality and concentration detection, 2 μg of total RNA was used to synthesize the first strand of cDNA with TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (Transgen, Beijing, China).Primers used for cDNA synthesis were random primer and anchored oligo (dT) 18 .The reaction was carried out by incubating the reaction mixture at 42 °C for 30 min, and then at 85 °C for 5 min.

Droplet digital PCR and Real-time quantitative PCR
We detected NPM1-mutated transcripts on real-time quantitative PCR (RT-qPCR) using a specific primer with a common primer and probe [23].
A PrimePCR TM ddPCR TM Mutation Detection Kit Assay (Bio-Rad Laboratories, Hercules, CA, USA) was used to detect an IDH2 p.R140Q mutant allele with genomic DNA according to the manufacturer's instructions.Analysis was performed using QuantaSoft Software (Bio-Rad Laboratories, Hercules, CA, USA).The sensitivity of ddPCR is declared by the manufacturer at a level of 0.1%.

Cell culture
All cell lines used in this study were purchased from ATCC (Manassas, VA, USA).HEK293T cells were cultured in Dulbecco's modified Eagle's medium with 15% fetal bovine serum (FBS, Gibco).The interleukin 3 (IL-3) dependent Ba/F3 or 32D cells were cultured in RPMI-1640 medium supplemented with 10% FBS (Gibco) and 10 ng/ml recombinant murine IL-3 (PeproTech, NJ, USA).All cell lines have been authenticated using STR profiling within 1 year.All experiments were performed with mycoplasmafree cells.

Retroviral transduction and murine BM transplant
HEK293T cells were transfected with the interested MSCV plasmids in the presence of FuGENE 6 (E2691, Promega, Madison, WI, USA) to produce packaged retroviruses according to the manufacturer's instructions.C-kit + cells from female C57BL/6 mice (Institute of Zoology, CAMS & PUMC) BM were isolated using CD117 isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and pre-stimulated for 12 h in StemSpan SFEM (StemCell Technologies, Vancouver, Canada) supplemented with 20 ng/ml mIL-3, 20 ng/ml mIL-6, and 50 ng/ml mouse stem cell factor (mSCF), then infected with retrovirus expressing SP or empty vector, respectively.A total of 16 lethally irradiated recipient male mice were intravenously transplanted with 1 × 10 6 GFP + c-kit + cells and the incidence of leukemia was observed.
Male mice used in the drug-treatment experiments were sublethally irradiated recipient mice.These mice were generated by intravenous inoculation of 5 × 10 5 GFP + spleen cells from the S7A4 mouse.The assignment of mice to each group was done randomly on day 5 after injection, and the specific number of mice in each group can be seen in Supplementary Fig. 3.The percentage of GFP + cells of peripheral blood and body weight were measured dynamically.The survival time of each group was recorded and compared.
Mice were maintained in specific pathogen-free (SPF)-certified facilities.Six-to eight-week-old mice were used in all experiments, which were approved by the Animal Care and Use Committee at the institution.
For Co-IP experiments, the HEK293T cells transiently transfected with pMSCV-SP-Flag-IRES-GFP and pMSCV-SP-Myc-IRES-GFP plasmids together were lysed with cell lysis buffer for western blotting and IP (Beyotime, Beijing, China).They were then pre-mixed with a protease inhibitor cocktail and PMSF on ice for 30 min and centrifuged at 12,000 rpm for 15 min.The supernatants were precleared using protein A/G-PLUS agarose beads (IP05; Millipore, MA, USA) for 1 h, followed by incubation with 2 ug of anti-Flagtag monoclonal antibodies (Sigma, F1804), anti-Myc-tag monoclonal antibody (71D10, #2278, 1:200; CST, USA), anti-rabbit or anti-mouse IgG (Cell Signaling Technology) in a total of 500 ul of lysis buffer for 2 h.The lysates were then incubated with 30 ul of protein A/G-agarose beads (IP05; Millipore, MA, USA) overnight at 4 °C.The beads were pelleted by centrifugation and washed three times with 500 ul of lysis buffer.The immunocomplexes were isolated and purified according to the manufacturer's protocol, and then were subjected to western blot analysis using the following primary antibodies: anti-Myc-tag monoclonal antibody from CST, anti-Flag-tag monoclonal antibodies from Sigma-Aldrich.Anti-β-actin monoclonal antibody (Cell Signaling Technology) was used as internal controls.
Flow cytometry assays were performed using ACEA NovoCyte® 3000 flow cytometer and NovoExpress software (ACEA) was used for data acquisition and analyses.Antibodies used in the present study are listed in supplementary Table 4.

Statistical analysis
Data were shown as mean ± standard deviation (SD).The significance of differences between the two groups was determined by the Student t-test.Data analyses were performed using GraphPad Prism 9.0 software.The survival time was measured by Kaplan-Meier method and the log-rank analysis.Statistical significance threshold was set at 0.05; asterisks indicate significant differences (*P < 0.05; **P < 0.01; and ***P < 0.001).
Due to the older age and pulmonary infection, he received a lower-intensity induction therapy comprising azacytidine and HA (homoharringtonine, cytarabine).After two cycles of induction therapy, the patient was assessed as having complete response (CR) with negative minimal residual disease (MRD) in FCM.Subsequently, the patient received two courses of sequential consolidation chemotherapy with intermediate-dose cytarabine (IDAC) regimen and imatinib as maintenance therapy.However, after 15 months from diagnosis, the patient suffered a relapse with an expansion of the NPM1 ancestral clone and high allelic ratio (AR) FLT3-ITD mutation.He then was administered venetoclaxazacitidine combined with sorafenib as re-induction therapy, which resulted in CR.The patient is still on regular maintenance therapy and follow-up for ongoing monitoring of his condition.Identification of t(5;14)(q32;q12) translocation and STRN3::PDGFRB fusion transcript in BM cells The BM cells were analyzed for chromosomal abnormalities, revealing a clonal translocation t(5;14)(q32;q12) in 2 out of 20 metaphases prior to the second induction therapy (Fig. 1B).FISH studies found the PDGFRB rearrangement in 15% of cells examined (Fig. 1C), which was above the laboratory-determined cutoff for a separated signal of 3.37%.

Subclonal acquisition of STRN3::PDGFRB fusion in an NPM1mut AML
The leukemic blasts exhibited morphologically and immunophenotypically homogeneous monocytic characteristics and the FISH showed 15% of examined cells with PDGFRB rearrangement.Therefore, we suspected that the STRN3::PDGFRB clone might be a subclone that emerged as a secondary event during the disease development rather than as the founding clone of AML.
from the FLT3-ITD (c.1754_1837dup) clone at diagnosis.In contrast, the IDH2 and NPM1 mutations remained stable with the persistence of the diagnosis mutations at relapse.Additionally, two distinct subclones with NUP50 or ZPBP2 mutations arose from the dominant clone with IDH2/NPM1/FLT3-ITD mutations (Fig. 3B).Our findings were further supported by data obtained from ddPCR and RT-qPCR, which were used to track the novel fusion gene clone and the major clone, and by examining the frequency of hotspot mutations and genomic lesions in 8 longitudinal samples obtained between diagnosis and relapse (Fig. 3C).

Oncogenic properties of STRN3::PDGFRB fusion gene and its transforming capabilities in vitro
To investigate the transforming capabilities of STRN3::PDGFRB, we designed a bicistronic MSCV-based retroviral plasmid containing STRN3::PDGFRB and eGFP, along with truncated mutation variants lacking different domains of the STRN3 portion of fusion protein (Fig. 4A).We compared these constructs to an empty vector carrying only eGFP and the previously described fusion oncogene ETV6::PDGFRB.The IL-3 dependent murine hematopoietic cell lines Ba/F3 and 32D were infected with these retroviral vectors, and after IL-3 removal, cells infected with the MSCV empty vector or the coiled-coil domain truncated mutant died (Fig. 4B, C).In contrast, cells infected with either STRN3::PDGFRB or ETV6::PDGFRB were IL-3 independent (Fig. 4B, C).The proliferation rate of STRN3::PDGFRB transduced cells were faster than that of ETV6::PDGFRB transduced cells without IL-3.When IL-3 was removed, the caveolin-binding site truncated mutant transduced cells proliferated slower than STRN3::PDGFRB transduced cells (Fig. 4B, C).The caveolin-binding site truncation reduced the transforming capabilities of the novel fusion oncoprotein but not as significantly as the coiled-coil domain, whereas the calmodulin-binding site truncation has less effect on the fusion protein.The expression of the fusion genes was confirmed by Western blotting (Fig. 4B, C).The IL-3 independence of these cells suggested the novel PDGFRB fusion oncogene has transforming properties.
To further understand the disease process, cells from the spleen were transplanted into sublethally irradiated secondary recipients and monitored weekly.We found that the Gr-1/ CD11b positive cells did not have a clonal advantage and disappeared gradually after transplant, but CD3 positive T cells gradually became dominant clone (Supplementary Fig. 1).These secondary recipients developed T-cell lymphoblastic lymphoma with a latency period of 4 to 6 weeks.In previous CML and CMML models [25,26], the disease could be serially passaged up to two rounds, and in all cases of serial passage, the myeloproliferative disease transforms into T-cell lymphomas.The secondary receipt mice were killed when symptomatic, and all lymph node groups were affected, as well as periportal tissue in the kidney, diffuse involvement of the spleen and liver, and of BM with lymphoblastic lymphoma (Fig. 6A, C).The Wright-Giemsa-stained PB smear showed a tumor with features of lymphoblastic leukemia, including intermediate-sized lymphoid cells with scant cytoplasm, and dispersed nuclear chromatin, (Fig. 6B).Western blot of spleen cells from different mice confirmed the expression of the STRN3::PDGFRB fusion protein (Supplementary Fig. 2).FCM from BM, spleen, blood, and lymph nodes showed that the leukemia cells are CD3 + , CD4 + , CD8 + , CD11b − , Gr-1 − cells (Fig. 6D).
Ba/F3 cells with STRN3::PDGFRB were sensitive to TKIs and XPO1 inhibitors, however, only imatinib alone could improve the survival of STRN3::PDGFRB tertiary recipient mice Ba/F3 cells expressing the STRN3::PDGFRB and ETV6::PDGFRB were significantly sensitive to TKIs, including imatinib, dasatinib, ponatinib, olverembatinib dimesylate, and XPO1 inhibitor selinexor (Fig. 7A-E).The efficacy of the combination of imatinib and selinexor was analyzed using an interactive analysis of multidrug combination profiling with the ZIP model, which showed a marked synergistic effect in STRN3::PDGFRB and ETV6::PDGFRB transduced Ba/F3 cells (Fig. 7F-H).The highest ZIP synergy scores were observed at concentrations of 10-50 nM, indicating that even low concentrations of imatinib and selinexor are effective in combination.
Previous studies have shown that imatinib can improve the survival of mice with ETV6::PDGFRB-induced malignancies [27,28].To evaluate the effect of imatinib on this model, S7A4's spleen cells were introduced into syngeneic recipients (Supplementary Fig. 3).The tertiary recipients were divided into groups that received daily doses of vehicle, selinexor (15 mg/kg, biw) alone, imatinib (100 mg/kg, qd) alone, or a combination of imatinib (75 mg/kg, qd), and selinexor (10 mg/kg, biw).Mice treated with imatinib alone had significantly prolonged survival over other groups, demonstrating that imatinib can inhibit the growth of tumor cells expressing STRN3::PDGFRB even after the transformation and is safe (Fig. 7I, J).Treatment of imatinib alone or in combination can obviously reduce leukemia burden in the spleen (Fig. 7I).However, treatment of tertiary recipients with selinexor (10 mg/kg twice per week or 15 mg/kg twice per week) was ineffective in prolonging survival (Fig. 7J) and caused serious gastrointestinal side effects.All mice treated with selinexor were emaciated (Mean weight: 22 to 16 g), and many of them appeared with abdominal distension and anorexia, indicating intestinal obstruction (Supplementary Fig. 4).The serious gastrointestinal side effects may significantly affected feeding and nutritional status, resulting in no increase in survival when the combination group compared with the control group (Fig. 7J).

DISCUSSION
Cancer arises through an evolutionary process of somatic mutation and selection.Although it may be depicted as a linear sequence of mutational events that produce a homogeneous cell population, tumor evolution is associated with significant intratumoral heterogeneity [29].Leukemia, like blood cancer, has a similar evolutionary process.Fusion genes generated as a consequence of reciprocal chromosomal translocations are commonly seen in hematologic malignancies, mainly as a driver mutation, and rarely appear in subclones as secondary events.Subclones containing some specific fusion genes may have survival advantages, which may occur at any time during the course and progress to a dominant clone, contributing to disease progression and/or relapse.BCR::ABL1 fusion gene, as a secondary event, have been reported in a series of AML or ALL cases [11,12,[30][31][32].Due to the small number of cases, the prognosis of AML with tyrosine kinase gene rearrangement subclones (AML-TKRS) is unknown.However, despite being a subclonal change in a few reported cases of AML, the presence of a BCR::ABL1 gene rearrangement did not seem to have a significant impact on prognosis, as several cases with co-occurring favorable mutations, such as RUNX1::RUNX1T1, CBFB::MYH11 or NPM1-mut, exhibited outcomes consistent with those expected for typical mutations [12,[31][32][33][34].Here we report a de novo NPM1-mut AML patient who had a subclone harboring the STRN3::PDGFRB fusion gene.The translocation (5q32; 14q12) and its derivative STRN3::PDGFRB fusion protein were identified for the first time.At the time of diagnosis, both FLT3-ITD and STRN3::PDGFRB subclones were detected in this patient (Fig. 3B).The clonal architecture of this patient is inferred based on NGS and ddPCR, not at a single cell level, thus giving rise to two possibilities: (1) IDH2 mutations were early genetic events that led to the generation of clonal hematopoiesis.NPM1 mutations were disease-defining genetic lesions that acted as gatekeepers for AML, whereas FLT3-ITD and STRN3::PDGFRB mutations were late events; (2) Under earlier mutations (such as IDH2), NPM1 and STRN3::PDGFRB subclones appeared simultaneously, with NPM1 as the predominant clone and FLT3-ITD subclone appearing later (Supplementary Fig. 5).
5 In vivo transforming activity of STRN3::PDGFRB.A Kaplan-Meier survival curves of recipient mice transplanted with different cells, the Mock and SP group indicate that lethally irradiated recipients receiving BM c-kit + cells transduced with empty vector or STRN3::PDGFRB cDNA, S7-F1 and S4-F1 group indicate that the secondary generation of sublethal recipients receiving spleen cells from S7 or S4, S7A4-F2 group indicate that the third generation of sublethal recipients receiving spleen cells from S7A4.B Representative picture of enlarged spleen from SP mouse (upper) compared with Mock mouse (lower).C Hematoxylin and eosin (H&E) stain showed extensive infiltration of leukemia cells in the bone marrow, lung, spleen, liver (scale bars: 50 μm in the BM, spleen, lung, 100 μm in the liver, upper: Mock group, lower: SP group).The BM and red pulp of the spleen in the SP group is replaced by sheets of granulocytes.D SP leukemia from peripheral blood can be defined as Gr1 Fig. 6 The secondary recipients developed T lymphoblastic lymphoma.A Representative pictures of enlarged lymph node (left) and kidney mass (right) from T-lymphoma transformation mice.B The lymphoblastic cells with scant cytoplasm dispersed nuclear chromatin with high nucleus-to-cytoplasm ratios were observed in the peripheral blood.C Histological analysis showed extensive infiltration of leukemia blasts in BM, spleen, liver, thymus, kidney, and lymph node (HE staining, scale bars: 50 μm in the BM, spleen, liver, 100 μm in the thymus and kidney).D The BM from second-generation mice showed that leukemia cells are CD3 + CD4 + CD8a + CD11b − Gr-1 − Ter119 − B220 − cells.
However, since the patient's leukemia cells were homogeneous monoblasts in morphology and immunology, and mice models were inconsistent with the AML phenotype, so the second possibility was less likely.Should the diagnosis be classified under the broad category of MLN-TK or AML with NPM1 mutation?The author believes that the diagnosis should be based on the dominant clone, but the treatment choice should consider the subclonal features.The patient's final diagnosis of NPM1-mut AML was based on homogeneous monoblasts in the BM and type D NPM1 mutation revealed by NGS [2].
The aberrant tyrosine kinase generated plays a role in cellular proliferation, and hence may result in a survival advantage for those leukemic cells containing the PDGFRB rearrangement.To combat this, TKIs have proven effective in eradicating the leukemia clone with tyrosine kinase rearrangement.It should be noted, however, that while many patients with this condition have been treated with TKIs as a part of their therapy, it is still unclear whether they can benefit from this approach.If TKIs are not included in therapy, there is a high risk for the TKR-positive subclones [12].Furthermore, if TKI monotherapy is used as maintenance treatment, there is an increased risk of relapse risk for TKR-negative clone [12,35,36], as reported in this paper.Therefore, we believe that if AML-TKRS patients are sensitive to traditional chemotherapy, it may not be necessary to add TKIs during induction therapy.However, appropriate maintenance therapy should be selected according to their genetic background to effectively control both TKR-negative and positive clones.
PDGFRB is a member of the type III receptor tyrosine kinase (RTK) family, but the chimeric proteins produced by various PDGFRB rearrangements cannot be activated by PDGF due to the lack of ligand binding domains.Instead, PDGFRB fusion proteins express the RTK kinase domain as the C-terminal fusion protein partner, which relies on the promoter of the gene encoding the N-terminal fusion protein for its expression.Each fusion protein contains oligomerization motifs that contribute to dimerization and constitutive activation of the PDGFRB kinase domain [37].Myeloid/lymphoid neoplasms with PDGFRB rearrangement are heterogeneous and may involve myeloid and/or lymphoid neoplasms that occur simultaneously or sequentially in the same patient, indicating a stem cell disorder with multilineage involvement and a different morphologic appearance in BM and lymph nodes [38][39][40].We demonstrate that the expression of STRN3::PDGFRB in primary mice hematopoietic cells induced an MDS/MPN-like disease characterized by leukocytosis with granulocyte predominance, anemia and thrombocytopenia, splenomegaly, extramedullary hematopoiesis in multiple organs including spleen and liver, and the presence and expression of the STRN3::PDGFRB in affected tissues.Sublethally irradiated secondary recipients from spleen cells of diseased mice gave rise to T lymphoblastic lymphoma phenotype in majority of cases, a phenomenon similar to that described in murine retroviral transduction models of BCR::ABL [26,41].And the T-cell blast transformation may be related to the progenitors losing their ability for multilineage reconstitution after serial rounds of transplantation and show an increasing bias towards T-cell repopulation [42].TKIs, such as imatinib, can specifically inhibit the kinase activity of ABL (including BCR::ABL1), PDGFR, and c-KIT.Patients with de novo or secondary (blast phase) acute leukemia/ lymphoblastic lymphoma bearing PDGFRA/B fusion genes can achieve durable complete hematological and molecular remission with imatinib monotherapy [43,44].Additionally, selinexor can inhibit BCR::ABL1 in CML-BC cells and increase the survival of BCR::ABL1 mice [45].We hypothesized that T-cell blast transformation generated in our model may respond to combination therapy with an XPO1 inhibitor and imatinib.However, contrary to previous works and in vitro experiments, selinexor plus imatinib was not superior to imatinib alone, and even the survival of the selinexor plus imatinib group was similar to that of the control group due to severe gastrointestinal reactions that affected the absorption of imatinib.
In summary, we identified an NPM1-mut AML patient with two subclones at diagnosis, one of which harbored a previously unknown STRN3::PDGFRB fusion gene.We treated the patient with an imatinib single drug (targeting STRN3::PDGFRB subclone) as maintenance therapy resulting in a good response.However, this therapeutic approach caused a change in the clone evolution, leading to the development of an FLT3-ITD subclone that became the dominant clone at relapse.This finding highlights the importance of selecting appropriate treatments to target different subclones with distinct genetic backgrounds to prevent relapse in AML patients.In vitro and in vivo experiments demonstrated that the novel fusion gene was sufficient to induce hematological malignancy and was sensitive to TKI therapy.Overall, these results emphasize the complexity of AML subclones and the need for personalized treatment strategies to address the diversity of genetic abnormalities and subclones in individual patients.Fig. 7 Effects of TKIs or XOP1 inhibitors in vitro and in vivo.A-E Drug sensitivity of Ba/F3 cells with STRN3::PDGFRB, ETV6::PDGFRB, empty vector upon imatinib, dasatinib, ponatinib, olverembatinib, and selinexor was detected by CCK8 assay.F-H The synergy maps of Ba/F3 cells with empty vector (F), ETV6::PDGFRB (G), STRN3::PDGFRB (H) treated with the indicated concentration of imatinib and/or selinexor for 72 h (n = 3), red regions indicate synergism; white-additive effect; green-antagonism.I Effects of imatinib (100 mg/kg) and/or selinexor (15 mg/kg) on the spleen size of T-cell lymphoma transformation mice.The T-cell lymphoma transformation models were intravenously injected with the spleens from the S7A4 mouse and then randomly divided into different groups.All animals were treated for 7 days and collected spleen to compare the leukemia burden.J Kaplan-Meier survival curves of T-cell lymphoma transformation mice treated with imatinib and/or selinexor.The mice began treatment after the BMT for 5 days until death.
rearrangement clone as a subclone at diagnosis

Fig. 1
Fig. 1 Laboratory data of the patient.A Patient's bone marrow aspirate smear using Wright-Giemsa stain.The monocyte blast with Auer rods (black arrow).Original magnification (×1000); B The G-banded karyotype showing the translocation t(5;14) (q32;q12).The red arrows indicate the structural aberrations of chromosomes 5 and 14; C The red and green separated signals were found in 15% of blast cells; D The data from RNA-seq showed that the STRN3 gene fused to the PDGFRB gene.

Fig. 3
Fig. 3 Clonal architecture of this patient.A Mutational landscape of leukemia cells at diagnosis, remission, and relapse.B A model of clonal evolution across disease progression.Two subclones within the founding clone appeared at diagnosis, including the STRN3::PDGFRB subclone and FLT3-ITD subclone.C The hotspot mutations burden at different points during follow-up.