IDH2 mutation accelerates TPO‐induced myelofibrosis with enhanced S100a8/a9 and NFκB signaling in vivo

Abstract Introduction IDH2 mutation is an unfavorable prognostic factor in patients with primary myelofibrosis (PMF) but its effect on myelofibrosis (MF) remains largely unclear. Methods In this study, we aimed to elucidate the roles of IDH2 mutation in the development and progression of MF by transcriptomic and molecular techniques using the Idh2 R172K transgenic mice. Results We found that thrombopoietin (TPO)‐overexpressed Idh2 R172K (Idh2 R172K + TPO) mice had accelerated progression to MF, compared with TPO‐overexpressed Idh2‐wild (WT + TPO) mice, showing activation of multiple inflammatory pathways, among which nuclear factor κB (NFκB) was the most significantly enhanced. Single‐cell transcriptomes of the marrow cells in early MF showed that S100a8/a9 expression was mainly confined to neutrophil progenitors in the WT + TPO mice, but highly expressed in several types of myeloid precursor cells, including the megakaryocyte progenitors in the Idh2 R172K + TPO group. Furthermore, Idh2 R172K mice at age of 18 months had larger spleens, increased S100a8/a9‐Tlr4 expression, and elevated serum S100a8/a9 levels compared with WT mice. PMF patients with IDH2 mutations had higher bone marrow plasma S100A8/A9 levels than those without IDH2 mutations. Conclusion Overall, our findings showed that IDH2 mutation induced proinflammatory effects, which further exacerbated MF, as evidenced by the increase in S100a8/a9 levels and NFκB hyperactivation in Idh2 R172K + TPO mice.

levels compared with WT mice.PMF patients with IDH2 mutations had higher bone marrow plasma S100A8/A9 levels than those without IDH2 mutations.

Conclusion:
Overall, our findings showed that IDH2 mutation induced proinflammatory effects, which further exacerbated MF, as evidenced by the increase in S100a8/a9 levels and NFκB hyperactivation in Idh2 R172K + TPO mice.

K E Y W O R D S
IDH2 mutation, myelofibrosis, S100a8/a9, TPO

INTRODUCTION
Philadelphia chromosome (Ph)-negative myeloproliferative neoplasms (MPNs) are rare hematologic disorders characterized by clonal expansion of myeloid cells, constitutional symptoms, and inherent risk of progression to myelofibrosis (MF) and leukemia [1].Classic MPNs consist of three subtypes: polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) [1].PMF is further divided into pre-PMF and overt-PMF according to the fibrosis severity in the bone marrow (BM).PV and ET patients are at risk of developing post-PV or post-ET secondary MF after a long period of time.Patients with MF have the worst prognosis and highest risk of acute myeloid leukemia (AML) transformation among classic MPNs [1,2].
MPNs arise from somatic mutations of three important and mutually exclusive driver genes: JAK2, the chaperone calreticulin (CALR), and the thrombopoietic receptor MPL in hematopoietic stem cells (HSC).
In addition to the well-known JAK-STAT pathway, activation of the nuclear factor κB (NFκB) pathway was recently identified as a vital mechanism involved in BM inflammation in MF [3][4][5].A recent study showed that compared with JAK inhibition alone, dual inhibition of JAK-STAT and NFκB pathways could more effectively prevent disease progression and might even reverse fibrosis in BM [4].NFκB signaling hyperactivation is induced by a combination of cell-autonomous and non-cell-autonomous pathways following JAK-STAT activation and cytokine stimulation, including tumor necrosis factor (TNF, also known as TNF-α), interleukin-1 (IL-1) and IL-6, and Toll-like receptor (TLR) ligands, such as the alarmin heterocomplex S100A8/A9 [6].Dysregulation of these pathways induces myeloproliferative disorder in BM in all MPN patients regardless of the mutation type [1].In MPNs, inflammatory cytokines are responsible for the highly deleterious pathophysiological process in the BM microenvironment, suggesting that MPN is "a human inflammation model for cancer development." However, MPN is considered more of an inflammatory disease than cancer [3,6,7].
Despite these data, the mechanism by which IDH2 mutations accelerate MF progression remains unknown.Therefore, this study aimed to elucidate the role of IDH2 mutation in the pathogenesis of MF using transcriptomic and molecular techniques.To achieve this, we induced MF in mice by overexpressing TPO in Idh2-wild (WT) and Idh2 R172K-mutated (Idh2 R172K ) HSC, followed by a detailed examination of hematopoietic phenotypes.

Generation of Idh2 R172K mice
Idh2 R172K transgenic mice were generated for the experiments as described in our previous study [20].Briefly, we introduced an AGG → AAG point mutation into exon 4 of the murine Idh2 locus, leading to the R172K mutation (Idh2 R172K ).Mice were housed in a specific pathogen-free animal facility, and the study was approved by the Institutional Animal Care and Use Committee (IACUC) of National Taiwan University College of Medicine (IACUC approval number: 20210425).

Retroviral transduction of TPO in mouse BM cells
WT and Idh2 R172K mice (8-12 weeks old) were intraperitoneally injected with 150 mg/kg 5-fluorouracil (Merck) 3 days before BM harvesting.Retrovirus constructs carrying TPO tagged with green fluorescence (GFP) were transfected into a Plat-E retroviral packaging cell line (Cell Biolabs) 2 days before viral transduction.On the day of viral transduction, the virus concentrate was applied to the bone marrow cells (BMC) harvested from donor mice cultured in medium with cytokines as previously described [21].

Retroviral transduction of WT IDH and IDH R172K in SET-2 cell line
The SET-2 cell line was purchased from DSMZ (ACC 608).A retrovirus construct carrying WT IDH2 or IDH R172K tagged with GFP was transfected into a Plat-A retroviral packaging cell line (Cell Biolabs) 2 days before viral transduction.The virus concentrate was applied to the SET-2 cell line cultured in RPMI medium containing 20% fetal bovine serum (FBS).

2.4
Fluorescence-activated cell sorting and analysis GFP-positive (GFP + ) cells were sorted and analyzed using a FACSLSRII (BD Bioscience)/Attune (Thermo Fisher) at the Flow Cytometric Analyzing and Sorting Core Facility at the National Taiwan University Hospital (NTUH).

BM transplantation assay
GFP + viable BMC were sorted 72 h after viral transduction and transplanted into lethally irradiated recipient mice as previously described [21].B6.SJL-Ptprc α pepc β / BoyJ (CD45.1)recipient mice received a single lethal dose of 10 Gy irradiation followed by retro-orbital injection of 100,000 GFP + donor cells along with 10 5 CD45.1 helper BMC.We checked the hemogram monthly after transplantation of the mice and sacrificed them at 12 weeks post-transplant for further histologic and transcriptomic studies.For those used to explore the single-cell transcriptomes of early MF, mice were sacrificed 4 weeks post-transplant.

Analysis of the gene expression profile
Raw reads were aligned to the mouse reference genome GRCm38, and gene-level expression was quantified in expected counts and transcripts per kilobase million (TPM) using RNA-Seq by Expectation Maximization (RSEM) software.The RSEM expected counts were normalized using the trimmed mean of the M-values method implemented in the R package edgeR.Differentially expressed genes (DEGs) were identified using the Wilcoxon rank sum test.Genes that were detected in more than 25% of a specific cluster were included in the DEG analysis, with the minimum fold-change threshold set to 1.25.Gene set enrichment analysis (GSEA) was implemented in the R package clus-terProfiler [22] and the gene sets used for analysis were downloaded from MSigDB (https://www.broadinstitute.org/gsea/msigdb/).Genes were pre-ranked in the order of differential expression between the conditions [21,23].Protein-protein interaction network analysis was performed using STRING database v11.5 (available at: http://stringdb.org/)[24].We filtered for the interactions belonging to Mus musculus to grow the interaction network and further refined it to include only those interactions connecting S100a8 and S100a9 with confidence scores >0.5.

Single-cell RNA sequencing data pre-processing and analysis
Single-cell RNA sequencing (scRNA-seq) library preparation and analysis were performed as previously described [20].Approximately 15,000 lineage-negative BM cells from mice were FACS sorted into 20 µL Dulbecco's Modified Eagle Medium (DMEM)/10% FBS and then loaded onto the 10× chromium controller, according to the manufacturer's instructions (Supporting Information).
Lineage assignment of cell clusters was performed by inspecting the expression patterns of canonical lineage-specific markers that have been previously reported in the literature on uniform manifold approximation and projection (UMAP) space [25,26,27].

PMF patient cohort
We extended the PMF patient cohort from the NTUH with clinical information, including the results of cytogenetics and mutations detected by targeted next-generation sequencing, as previously mentioned [11,28].Institutional Review Board (IRB) of NTUH approved the collection of patients' BM samples for further studies (IRB approval number: 201709072RINC) with informed consent from all subjects.

Statistical analysis
Data were processed using GraphPad Prism (GraphPad Software) or Microsoft Excel (Microsoft), and differences between means were determined using Student's t-test.Means were considered significant at p < 0.05.

Idh2 R172K mutation accelerates MF progression in the TPO mouse model
Thrombopoiesis was induced in WT and Idh2 R172K transgenic mice [20] to evaluate the effects of IDH2 mutations in MF [29][30][31].We overexpressed TPO in WT and Idh2 R172K mouse marrow cells, followed by transplantation into syngeneic mice (designated as WT + TPO and Idh2 R172K + TPO hereafter).Idh2 R172K + TPO mice had larger spleens (Figure 1A,B), lower hemoglobin levels (p < 0.01), and higher white cell counts (p < 0.05) than WT + TPO mice 12 weeks after transplantation; however, there was no significant difference in platelet count between the groups (Figure 1C-E positive staining of collagen in the Idh2 R172K + TPO mice (Figure 1H), with some mice developing prominent osteosclerosis with increased formation and thickening of the trabecular bones (Figure 1H).

NFκB pathways were hyperactivated in young Idh2 R172K + TPO mice
The transcriptomes of whole BM cells from three pairs of WT + TPO and Idh2 R172K + TPO mice sacrificed at 12 weeks posttransplantation were compared to elucidate the mechanism that IDH2 mutation enhanced MF.Multidimensional scaling revealed distinct global gene expression patterns between the two groups of mice (Figure S1).GSEA revealed that several inflammatory signaling pathways were significantly upregulated in the Idh2 R172K + TPO group, including TNFα signaling via NFκB, interferon gamma response, and IL2_stat5, PI3K_Akt_Mtor, and IL6_Jak_Stat3 signaling (Figure 2A).
Notably, NFκB pathways, including TNFα signaling via the NFκB canon-ical pathway, were markedly upregulated in the Idh2 R172K + TPO group (Figure 2B,C).BM sections were IHC stained for phospho-p65 expression, a component of the activated NFκB complex, to confirm the functional activation of NFκB in Idh2 R172K + TPO mice.Consistent with the transcriptome data, phospho-p65 was highly expressed in the nuclei of multiple cell types, including hematopoietic stem and progenitor cells (HSPC), megakaryocytes, and other myeloid cells of the BM of Idh2 R172K + TPO mouse compared with the WT + TPO mice (Figure 2D).

NFκB pathways were hyperactivated in IDH2 R172K -transduced SET-2 cells
To further examine the direct effects of IDH2 mutation on megakaryocytic progenitors, we overexpressed WT IDH2 and IDH2 R172K in SET-2 cell line, a human megakaryoblast cell line originating from an ET

3.4
Hyperactivated S100a8/a9 expression and NFκB signaling in Idh2 R172K + TPO myeloid progenitors during early MF scRNA-seq of pre-fibrotic Lin − BM cells from mice (4 weeks posttransplantation) was performed to further elucidate the pathogenesis of MF.We focused on myeloid lineages and projected the cells onto the UMAP after removing batch effects and aligning cells from two pairs of samples (WT + TPO vs. Idh2 R172K + TPO).Cell types included myeloblast, multipotent progenitor, myeloid progenitor (Mye_Prog), megakaryocyte progenitor (Meg_Prog), erythroid progen-itor (Ery_Prog), erythroid cell (Ery), neutrophil progenitor (Neu_Prog), and various subpopulations of monocytes and dendritic cells.Compared with WT + TPO group, the Idh2 R172K + TPO group had higher percentages of HSPC, Meg_Prog, and neutrophils, but a lower percentage of erythroid cells (Figure 3A), suggesting a more advanced disease stage [33].The inflammatory alarmins S100a8 and S100a9 were among the top DEGs (Table S1) in various myeloid cells in Idh2 R172K + TPO mice, including mature and immature populations.In contrast, their expression was confined to neutrophils and neutrophil progenitors in WT + TPO mice (Figure 3B,C).
Since megakaryocytes are critical for MF progression [34], we focused on the Meg_Prog population.There was a significant increase in S100a8 and S100a9 expression in Meg_Prog in the Idh2 R172K +TPO group compared with the WT + TPO group (Figure 3D,E).Moreover, GSEA indicated that the NFκB pathway was significantly enriched in the Idh2 R172K + TPO group (Figure 3F), indicating the importance of S100a8/a9 and NFκB in MF progression in Idh2 R172K mice.Furthermore, IHC staining confirmed higher S100a8 expression in the BM sections of Idh2 R172K + TPO mice (Figure 3G).

F I G U R E 4
Aged Idh2 R172K mice had higher alarmin S100a8/a9 expression and inflammaging phenotypes compared with age-matched littermates.A Idh2 R172K mice at 18 months old had larger spleens compared with age-matched wild-type (WT) littermates.Upper: WT (n = 4); lower: Idh2 R172K (n = 5).(B) Determination of the spleen weight of mice in the two groups: WT (n = 4); Idh2 R172K (n = 5).(C) Serum S100a8/a9 levels were not significantly different between young WT and Idh2 R172K mice; however, aged Idh2 R172K mice had significantly higher levels than their littermate controls (n = 3 in each group).(D) Expression levels of S100a family genes.Several S100a genes, including S100a10, S100a8, S100a9, S100a1, and S100a6 were upregulated in the Idh2 R172K group compared with the control group (n = 3 in each group), illustrated as heatmap.S100a8 (E) and S100a9 (F) expression levels were significantly higher in Idh2 R172K mice compared with the control group (n = 3 in each group).(G) Protein interaction network analysis using STRING database specifically connected S100a8 and S100a9.The S100a8/a9 receptor Tlr4 was highly connected with S100a8 and S100a9 in the dataset, suggesting an activation of the S100a8/a9-Tlr4 pathway in elderly Idh2 R172K mice.(H) Tlr4 expression was significantly higher in Idh2 R172K mice compared with the control group (n = 3 in each group).(I-K) Gene set enrichment analysis (GSEA) showed that pathways related with innate immunity were significantly upregulated, whereas hematopoietic stem and progenitor cell (HSPC) hematopoiesis pathways were downregulated (all p < 0.01), suggesting a proinflammatory status in aged Idh2 R172K mice.Significant differences were determined using a t-test; *p < 0.05.

3.5
Enhanced S100a8/a9 expression and inflammaging pathways in aged Idh2 R172K mice PMF is an inflammatory disease prevalent in the elderly.Aging has been shown to trigger sterile inflammation (inflammaging), which involves NFκB and several innate immune pathways [35,36].Although there were no marked difference in terms of hemogram and BM histologic findings between the aged Idh2 R172K and WT mice [20], significantly enlarged spleens were observed in aged (18 months) Idh2 R172K mice compared with WT littermates (Figure 4A,B).Additionally, serum S100a8/a9 levels were significantly elevated in aged Idh2 R172K mice compared with their littermate WT controls as well (Figure 4C).Consistent with these findings, several S100 family genes, including S100a10, S100a8, S100a9, S100a1, and S100a6, were significantly upregulated in whole BM cells from aged Idh2 R172K mice (Figure 4D-F).Of note, Tlr4, which encodes a TLR Tlr4 protein as the S100a8/9 receptor, was significantly upregulated in the Idh2 R172K mice (Figure 4G,H), indicating the activation of S100a8/a9 and TLR signaling pathways in Idh2 R172K mice during aging.GSEA showed that several immune-related pathways were activated, whereas HSC pathways were downregulated in aged Idh2 R172K mice compared with aged WT mice.Overall, these data indicate a proinflammatory function for Idh2 R172K mutation (Figure 4I-K).

S100A8/A9 levels in BM plasma were higher in IDH2-mutated MF patients
From the findings in the mouse model of TPO-induced MF shown above, it was suggested that Idh2 mutation might increase S100A8/A9 levels.To verify this in humans, the S100A8/A9 levels of the BM plasma obtained from MF patients were determined using ELISA kit.Among the 36 patients with available BM plasma S100A8/A9 levels and targeted sequencing data, two harbored IDH2 mutations with similar F I G U R E 5 Higher expression of S100A8/A9 heterodimer in the bone marrow (BM) of IDH2-mutated primary myelofibrosis (PMF) patients.(A) Mutation landscape in PMF patients.The profiles were sorted according to plasma S100A8/A9 expression (top bar), which was determined using enzyme-linked immunosorbent assay (ELISA) kits (left to right, low to high).Driver mutations, blue color; high molecular risk (HMR) mutations, dark green color; IDH2 mutation, yellow color; other mutations, light green color.(B) S100A8/A9 expression levels between PMF patients with (n = 2) and without (n = 34) IDH2 mutation was illustrated using dot plots.S100A8/A9 expression was significantly higher in patients with IDH2 mutation.(C) Graphic description of the summary of this study.Significant differences were determined using a t-test; *p < 0.05.allele frequencies at 48.4% and 49.4%, respectively.The mutation profiles of the patients and their corresponding S100A8/A9 levels are shown in Figure 5A.The two IDH2-mutated BM samples had significantly higher S100A8/9 expression levels than the other samples (Figure 5B).Overall, our study demonstrated that Idh2 R172K -mutated BM cells produced more S100a8/a9 alarmin (a TLR4 ligand) during aging and JAK-STAT activation, leading to the hyperactivation of NFκB pathway and more severe MF phenotypes in a TPO mouse model.A schematic illustration of these findings is shown in Figure 5C.

DISCUSSION
In MPN, hyperactivation of the JAK-STAT pathway induces the proliferation of myeloid lineage cells and cytokines production, including IL-1, IL-6, IL-8, and TNF, and the alarmin heterocomplex S100A8/A9 [6,34].Concurrent mutations other than the three major driver mutations (JAK2, CALR, MPL mutations) of JAK-STAT pathway are associated with disease progression, including MF and AML transformation.Specifically, IDH mutations have been recognized as a poor prognostic factor of MF [11,37,38].Moreover, IDH2 mutations were identified as the only high-risk mutations in ET, PV, and PMF in the Mayo-Florence cohorts [10,13].Although IDH mutations are clinically relevant, their role in the pathogenesis of MPN remains unclear.McKenney et al.
showed that mice with combined Jak2 V617F and IDH mutations had higher levels of HSPC, and treatment with combined inhibitors normalized the HSPC levels and reversed aberrant gene expression in murine models.However, both double-hit and single JAK2V617F mutant mice exhibited similar phenotypes [39].In the present study, MF progression through a self-perpetuating cycle of the inflammation network between stromal cells and hematopoietic cells [34].
The impact of IDH mutations on the cytokine signaling response in HSPC remains largely unknown.In the present study, scRNA-seq analysis showed that S100a8/a9 and NFκB signaling were significantly upregulated in TPO-overexpressed Idh2-mutated BM HSPC and megakaryocyte progenitors compared with the Idh2-wild counterpart.
Moreover, S100a8/a9 levels were also elevated in aged Idh2-mutated mice, which is consistent with a sterile inflammatory phenotype.Additionally, transcriptome analysis revealed positively enriched innate immune pathways and negatively enriched HSPC pathways in aged Idh2-mutated mice, which are the two hallmarks of inflammaging.
These results are consistent with a recent report, in which IDH2mutated leukemia cells were highly sensitive to IL1β, with augmented response at multiple downstream targets of the NFκB pathway [49].
That study showed that IDH2-mutated AML cell lines exhibited significantly higher sensitivity to IL-1β across various downstream readouts.
Additionally, bulk RNA sequencing revealed elevated cytokine-related signaling pathways and NFκB target genes.scRNA-seq of both unstimulated and stimulated cells confirmed modified IL-1β transcriptional responses in the IDH2-mutant cells.Targeted inhibition of the IκB kinase complex reduced IL-1β responses and induced cell death in primary IDH-mutated leukemia samples [49].
Recent studies have shown that MSCs in the BM also play important inflammatory roles in the BM [34].In IDH-mutated AML, 2HG activates the NFκB pathway in BM stromal cells, leading to the production of cytokines, including IL-6, IL-8, and complement 5a, which might stimulate the progression of AML [50].The pathological roles of BM stromal cells in IDH-mutated MPN need to be explored in the future.In summary, our study revealed the important roles of the alarmin heterocomplex S100A8/A9 in Idh2-mutated mice during aging and early MF formation, providing new insights for further mechanistic and therapeutic exploration.

F I G U R E 1
Idh2 R172K + thrombopoietin (TPO) mice had more severe myelofibrosis (MF) phenotypes than wild-type (WT) + TPO mice.(A) Representative images of the spleens of mice sacrificed at 12 weeks post-transplant.Upper: WT + TPO (n = 5); lower: Idh2 R172K + TPO (n = 5).(B) Spleen weight of mice in the two groups (n = 5 in each group).(C-E) Hemogram of the two groups of mice at 12 weeks post-transplant: (C) white blood cells (WBC), (D) hemoglobin, and (E) platelets (n = 5 in each group).Idh2 R172K + TPO mice had significantly higher WBC counts and lower hemoglobin levels than the control group.(F) Bone marrow (BM) sections showed increased megakaryocytes in both groups, consistent with the TPO-related myeloproliferative neoplasm (MPN) phenotype.Cellular streaming (green arrow) and dilated sinusoids with intra-sinusoidal hematopoiesis (yellow arrow) were noted in the Idh2 R172K + TPO group, implying a more severe MF phenotype (original magnification ×100).(G) Reticulin staining revealed significantly increased reticulin in the Idh2 R172K + TPO group (grade 3) compared with the WT + TPO group (grade 1) (original magnification ×100).(H) Masson's trichrome staining showed collagenous fibers and significant osteosclerosis in the Idh2 R172K + TPO group compared with the control group (original magnification ×100).Significant differences were determined using a t-test; *p < 0.05, **p < 0.01.

F I G U R E 3
Single-cell transcriptome profiling of bone marrow cells (BMCs) from Idh2-wild (WT) + thrombopoietin (TPO) and Idh2 R172K + TPO mice in earlier stage of myelofibrosis (MF) formation (4 weeks post-transplant).(A) Uniform manifold approximation and projection (UMAP) representation of single cells from the WT + TPO and Idh2 R172K + TPO mice Lin-BMC, classified into 12 distinct clusters.Cells were annotated by the expression pattern of canonical lineage-specific markers, according to previously described methods.(B) S100a8 expression levels in various subpopulations.(C) S100a9 expression levels in various subpopulations.(D) Violin plots of normalized expression of S100a8 in Meg_prog.The level was significantly higher in the Idh2 R172K + TPO group (p < 0.001).(E) Violin plots illustration of normalized expression of S100a9 in Meg_prog.The level was significantly higher in the Idh2 R172K + TPO group (p < 0.001).(F) Gene set enrichment analysis (GSEA) showed upregulation of tumor necrosis factor (TNF-α) signaling via nuclear factor κB (NFκB) signaling (p < 0.001) in Meg_prog cells in Idh2 R172K + TPO mice.(G) Immunohistochemistry (IHC) staining revealed an increase in S100a8 expression in the Idh2 R172K + TPO group (original magnification ×100).patient harboring an inherent JAK2V617F mutation [32].Transcriptome analysis revealed that NFκB signaling was hyperactivated in IDH2 R172K -transfected SET-2 cells compared with cells transfected with WT IDH2 and empty vectors (Figure 2E,F), indicating that IDH2 mutation can activate NFκB under the background of JAK-STAT pathway activation.
-Chin Lin wrote the manuscript, designed the study, performed the experiments, and analyzed the data.Chi-Yuan Yao, Yu-Hung Wang, and Chia-Lang Hsu performed the bioinformatics analyses.Chang-Tsu Yuan interpreted tissue sections.Sze-Hwei Lee, Yueh-Chwen Hsu, Tsung-Chih Chen, Jhih-Yi Lee, Pin-Tsen Shih, Chein-Jun Kao, Po-Han Chuang, and Yuan-Yeh Kuo performed experiments.Hsin-An Hou performed the clinical data collection.Hwei-Fang Tien organized and revised the manuscript.Wen-Chien Chou provided the concept, coordinated the study, and revised the manuscript accordingly.
We believe that the phenotypic discrepancies between the study by McKenney et al. and ours are mainly due to the different murine MF models used.All mice induced by TPO developed dominant MF within a few months [29, 40], while for McKenney et al., the phenotypes of the JAK2V617F mutant knock-in mice are much more indolent TPO-overexpressed Idh2 R172K mice exhibited severe MF, compared to TPO-overexpressed Idh2-wild mice, which was partially related to the hyperactivation of the NFκB pathway, as revealed by RNA-seq and IHC staining of BM.