Sorafenib promotes graft-versus-leukemia activity in mice and humans through IL-15 production in FLT3-ITD-mutant leukemia cells

Abstract

Individuals with acute myeloid leukemia (AML) harboring an internal tandem duplication (ITD) in the gene encoding Fms-related tyrosine kinase 3 (FLT3) who relapse after allogeneic hematopoietic cell transplantation (allo-HCT) have a 1-year survival rate below 20%. We observed that sorafenib, a multitargeted tyrosine kinase inhibitor, increased IL-15 production by FLT3-ITD⁺ leukemia cells. This synergized with the allogeneic CD8⁺ T cell response, leading to long-term survival in six mouse models of FLT3-ITD⁺ AML. Sorafenib-related IL-15 production caused an increase in CD8⁺CD107a⁺IFN-γ⁺ T cells with features of longevity (high levels of Bcl-2 and reduced PD-1 levels), which eradicated leukemia in secondary recipients. Mechanistically, sorafenib reduced expression of the transcription factor ATF4, thereby blocking negative regulation of interferon regulatory factor 7 (IRF7) activation, which enhanced IL-15 transcription. Both IRF7 knockdown and ATF4 overexpression in leukemia cells antagonized sorafenib-induced IL-15 production in vitro. Human FLT3-ITD⁺ AML cells obtained from sorafenib responders following sorafenib therapy showed increased levels of IL-15, phosphorylated IRF7, and a transcriptionally active IRF7 chromatin state. The mitochondrial spare respiratory capacity and glycolytic capacity of CD8⁺ T cells increased upon sorafenib treatment in sorafenib responders but not in nonresponders. Our findings indicate that the synergism of T cells and sorafenib is mediated via reduced ATF4 expression, causing activation of the IRF7-IL-15 axis in leukemia cells and thereby leading to metabolic reprogramming of leukemia-reactive T cells in humans. Therefore, sorafenib treatment has the potential to contribute to an immune-mediated cure of FLT3-ITD-mutant AML relapse, an otherwise fatal complication after allo-HCT.

Figure 1. Sorafenib synergizes with allogeneic T-cells and improves survival in FLT3-ITD driven AML mouse models by increasing IL-15 production

(a) Percentage survival of C57BL/6 recipient mice receiving AML^{MLL-PTD FLT3-ITD} cells (C57BL/6 background) and BALB/c BM or C57BL/6 BM (syn BM) with or without additional BALB/c T-cells (allo Tc) or C57BL/6 T-cells (syn Tc) and treated with either vehicle or sorafenib. The experiment was performed twice and the results were pooled; n=12 biologically independent animals per group are shown, except for the group Syn BM+AML^{MLL-PTD FLT3-ITD}+ Sorafenib+Syn Tc, here n=8 biologically independent animals per group are shown. P-values were calculated using the two-sided Mantel-Cox test. (b) Percentage survival of BALB/c recipients receiving C57BL/6 BM and Ba/F3-ITD cells with or without additional C57BL/6 T-cells and treated with vehicle or sorafenib as described in (a). The experiment was performed three times and the results were pooled; BM+Ba/F3-ITD+Vehicle (n=17, biologically independent animals), BM+Ba/F3-ITD+Tc+Vehicle (n=17, biologically independent animals), BM+Ba/F3-ITD+Sorafenib (n=19, biologically independent animals), BM+Ba/F3-ITD+Tc+Sorafenib (n=16, biologically independent animals), BM+Vehicle (n=4, biologically independent animals). P-values were calculated using the two-sided Mantel-Cox test. (c) Bioluminescence imaging (BLI) on day 10 after Ba/F3-ITD^luc cell transplantation showing the expansion of Ba/F3-ITD^luc cells in BALB/c recipients transplanted with C57BL/6 BM and Tc and, treated with vehicle or sorafenib. Shown are BLI images of a representative mouse from the 2 indicated groups. (d, e) Percentages of Ba/F3-ITD cells in the spleens of BALB/c mice on day 14, transplanted with C57BL/6 BM and Ba/F3-ITD cells with additional C57BL/6 T-cells, and treated with vehicle or sorafenib. (d) Percentage of GFP⁺Ba/F3-ITD cells in the spleens from each group. The experiment was performed three times and the results (mean ± s.e.m) were pooled; Tc+Vehicle (n=9, biologically independent animals per group), Tc+Sorafenib (n=10, biologically independent animals per group). The P-values were calculated using the two-sided Student’s unpaired t-test. (e) Representative flow cytometry plots showing the percentages of GFP⁺Ba/F3-ITD cells in the spleens of mice from each group. The data are representative of one experiment out of three independent experiments. (f, g) Microarray-based analysis of the expression levels of genes in Ba/F3-ITD cells that were treated with sorafenib (10 nM) or DMSO alone for 24 hours. (f) Tile display for the 25 most significantly regulated genes indicated by Robust Multichip Average (RMA) signal values, n=6, biologically independent samples per group. (g) Scatter plot showing the RMA values of Il-15 in Ba/F3-ITD cells, n=6, biologically independent samples per group. The P-values were calculated using two-sided Student’s unpaired t-test. (h) Quantification of Il-15 mRNA (mean ± s.e.m.) by qPCR in Ba/F3-ITD cells treated with 10nM sorafenib/DMSO relative to Mon2 mRNA. The experiment was performed three times and the results (mean ± s.e.m) were pooled, n=6 biologically independent samples per group. The P-values were calculated using the two-sided Student’s unpaired t-test. (i) The scatter plot shows the quantification of intracellular IL-15 (fold change of Mean Fluorescence Intensity (MFI) of IL-15 with respect to mean MFI of IL-15 from DMSO treated controls). As indicated Ba/F3-ITD cells (sorafenib sensitive) and Ba/F3-ITD F691L cells (harboring a FLT3 resistance mutation) were studied. The experiment was performed three times and the results (mean ± s.e.m.) were pooled; n=12 biologically independent samples per group, each data point represents a biologically independent sample. The P-values were calculated using the two-sided Student’s unpaired t-test. (j) Quantification of serum IL-15/IL-15Rα (mean ± s.e.m.) from naive BALB/c mice (n=5, biologically independent samples) or BALB/c recipients transplanted with C57BL/6 BM and Ba/F3-ITD cells with additional C57BL/6 T-cells and treated with vehicle (n=10, biologically independent samples) or sorafenib on day 14 (n=12, biologically independent samples) or day 22 (n=7, biologically independent samples) or day 98 (n=2, biologically independent samples) following Ba/F3-ITD cells injection. The dotted line represents the detection limit (4 pg/ml) of mouse IL-15/IL-15Rα ELISA. The experiment was repeated three times (except for the day 98 group) and the results (mean ± s.e.m.) were pooled. Each data point represents a biologically independent sample derived from an individual mouse. The P-values were calculated using a two-sided Mann-Whitney U test.

Figure 2. Sorafenib induced IL-15 production is derived from leukemia cells in vivo and synergizes with T cells in humanized mouse models

(a) The survival rate of C57BL/6 recipient mice is shown. Mice (C57BL/6) were transplanted with WT BALB/c BM, as well as with GFP⁺FLT3-ITD⁺ C57BL/6 BM to induce the leukemia. On day 2 T-cells (BALB/c) were given to induce the allogeneic immune effect. The GFP⁺FLT3-ITD⁺ BM was derived from either WT C57BL/6 mice (white open squares; WT leukemia no T-cells) (n=10, biologically independent animals) or from Il-15^−/− C57BL/6 mice to generate IL-15 deficient leukemia cells when indicated. In one group WT C57BL/6 recipients were transplanted with BALB/c BM, GFP⁺FLT3-ITD⁺ WT C57BL/6 BM and BALB/c T-cells and treated with sorafenib (red squares, BM/Tc WT leukemia sorafenib) (n=10, biologically independent animals). In another group Il-15^−/− C57BL/6 recipients were transplanted with BALB/c BM, FLT3-ITD⁺ WT C57BL/6 BM and BALB/c T-cells and treated with sorafenib (grey squares BM/Tc Il-15^−/− recipients + sorafenib) (n=10, biologically independent animals). In another group WT C57BL/6 recipients were transplanted with BALB/c BM, GFP⁺FLT3-ITD⁺ Il-15^−/− C57BL/6 BM and BALB/c T-cells and treated with sorafenib (black squares; BM/Tc Il-15^−/− leukemia + sorafenib) (n=11, biologically independent animals). In an additional setting WT C57BL/6 recipients were transplanted with BALB/c BM, GFP⁺FLT3-ITD⁺ Il-15^−/− C57BL/6 BM and BALB/c T-cells, and treated with sorafenib and IL-15 (green squares; BM/Tc Il-15^−/− leukemia, sorafenib + IL-15) (n=10, biologically independent animals). Biologically independent animals per group are shown. The experiment was performed twice and the results were pooled. P-values were calculated using the two-sided Mantel-Cox test. (b) Percentage of GFP⁺Ba/F3-ITD cells of all leukocytes in the blood of mice on day 14 from the groups described in (a). The experiment was performed twice and the results (mean ± s.e.m) were pooled; WT leukemia no T-cells (n=5, biologically independent samples), BM/Tc WT leukemia + sorafenib (n=5, biologically independent samples), BM/Tc Il-15^−/− recipients + sorafenib (n=5, biologically independent samples), BM/Tc Il-15^−/− leukemia + sorafenib (n=5, biologically independent samples), BM/Tc Il-15^−/− leukemia + sorafenib + IL-15 (n=7, biologically independent samples). The P-values were calculated using the two-sided Mann-Whitney U test and are indicated in the graph. (c) The scatter plot shows the histopathological scores from different GvHD target organs isolated on day 10 following allo-HCT of BALB/c mice transplanted with T-cells/vehicle or T-cells/sorafenib, or of C57BL/6 recipients transplanted with FLT3-ITD⁺ BM cells/T-cells/sorafenib/IL-15. The experiment was performed twice and the results (mean ± s.e.m.) were pooled; BM (n=10, biologically independent samples), BM+Tc+vehicle (n=20, biologically independent samples), BM+Tc+sorafenib (n=17, biologically independent samples), BM+il15^−/− leukemia+Tc+sorafenib (n=7, biologically independent samples). The P-value was calculated using the two-sided Mann-Whitney U test; P>0.05, non-significant (NS). (d) Percentage survival of BALB/c recipients receiving C57BL/6 BM and Ba/F3-ITD cells with additional C57BL/6 Tc and treated with sorafenib and non-specific IgG or anti-IL-15 antibody, as indicated for each group. The experiment was performed three times and the results were pooled, n=15 biologically independent animals per group. P-values were calculated using the two-sided Mantel-Cox test. (e) Percentage of GFP⁺Ba/F3-ITD cells of all living cells in the spleens on day 14 of mice from the groups as described in (d) with an additional group which was treated with vehicle. The experiment was performed two times and the results (mean ± s.e.m) were pooled; Tc+Vehicle (n=8, biologically independent samples), Tc+Sorafenib+isotype IgG (n=7, biologically independent samples), Tc+Sorafenib+anti-IL-15 (n=7, biologically independent samples). The P-values were calculated using the two-sided Mann-Whitney U test. P-values are indicated in the graph. (f) The survival rate of BALB/c recipients injected with Ba/F3-ITD cells is shown. Mice were transplanted with C57BL/6 WT BM (day 0) and on day 2 with additional C57BL/6 WT T-cells or Il-15Rα^−/− T-cells. The experiment was performed twice with similar results; n=12 biologically independent animals per group. Biologically independent animals per group are shown. P-values were calculated using the two-sided Mantel-Cox test. (g) Percentage survival of NSG mice receiving primary human FLT3-ITD⁺ AML cells from a HLA-A2⁺ patient with additional allogeneic human CD8⁺ T-cells that had been stimulated and expanded in the presence of autologous DCs expressing allogeneic HLA-A2 upon RNA transfection in vitro. Mice were treated with vehicle or sorafenib as indicated. The experiment was performed twice with similar results; n=8 biologically independent animals per group except for AML+ Vehicle (n=7 biologically independent animals). Biologically independent animals per group are shown. P-values were calculated using the two-sided Mantel-Cox test. (h) Percentage survival of Rag2^−/−Il2rγ^−/−recipient mice receiving human MV4-11 FLT3-ITD mutant) cells with or without additional C57BL/6 T-cells and being treated with vehicle or sorafenib. The experiment was performed three times and the results were pooled; n=9 biologically independent animals per group. Biologically independent animals per group are shown. P-values were calculated using the two-sided Mantel-Cox test. (i) Percentage survival of Rag2^−/−Il2rγ^−/−recipient mice receiving human MOLM-13 (FLT3-ITD mutant) cells with or without additional C57BL/6 T-cells and being treated with vehicle or sorafenib. The experiment was performed once, n=10 biologically independent animals per group. Biologically independent animals per group are shown. P-values were calculated using the two-sided Mantel-Cox test.

Figure 3. Sorafenib promotes cytotoxicity and longevity of donor CD8⁺ T-cells via IL-15

(a–d) Flow cytometry analysis of the spleens of BALB/c mice transplanted with C57BL/6 BM, Ba/F3-ITD cells (day 0) and T-cells (C57BL/6, day 2) as described in Figure 1b. The time point of analysis is day 12 following Ba/F3-ITD injection. (a) Scatter plot showing the fold change of MFI (with respect to mean MFI of vehicle treated group) for CD107a of all living donor-derived (H-2kb⁺) CD8⁺ T-cells from BMT recipients treated with vehicle (n=12, biologically independent samples) or sorafenib (n=15, biologically independent samples) or from untreated naive C57BL/6 mice (n=6, biologically independent samples) as indicated. The experiment was repeated three times and the results (mean ± s.e.m.) were pooled. The P-values were calculated using the two-sided Student’s unpaired t-test. (b) Scatter plot showing the fold change of MFI (with respect to mean MFI of vehicle treated group) for IFN-γ of all living donor-derived (H-2kb⁺) CD8⁺ T-cells from BMT recipients treated with vehicle (n=12, biologically independent samples) or sorafenib (n=12, biologically independent samples) or from untreated naive C57BL/6 mice (n=6, biologically independent samples) as indicated. The experiment was done three times and the results (mean ± s.e.m.) were pooled. The P-values were calculated by two-sided Student’s unpaired t-test; P>0.05, Not significant (NS). (c) Scatter plot showing the fold change of MFI (with respect to mean MFI of vehicle treated group) for CD40L of all living donor-derived (H-2kb⁺) CD8⁺ T-cells from BMT recipients treated vehicle (n=10, biologically independent samples) or sorafenib (n=12, biologically independent samples) or from untreated naive C57BL/6 mice (n=6, biologically independent samples) as indicated. The experiment was repeated three times and the results (mean ± s.e.m.) were pooled. The P-values were calculated using a two-sided Student’s unpaired t-test. (d) Scatter plot showing the quantification of phospho-STAT5 expression levels (the fold change of MFI with respect to mean MFI of vehicle treated group) in all living donor-derived (H-2kb⁺) CD8⁺ T-cells from BMT recipients treated with vehicle (n=16, biologically independent samples) or sorafenib (n=12, biologically independent samples) or from untreated naive C57BL/6 mice (n=6, biologically independent samples) as indicated. Each data point represents an individual sample of one biologically independent animal. The experiment was repeated three times and the results (mean ± s.e.m.) were pooled. The P-values were calculated with the two-sided Student’s unpaired t-test. (e) Survival rate of BALB/c mice which received Ba/F3-ITD cells, CD8⁺ Tc-depleted C57BL/6 BM and CD4⁺ T-cells; or Ba/F3-ITD cells, NK1.1-depleted C57BL/6 BM and T-cells and being treated with either sorafenib or vehicle. The experiment was repeated twice and the results were pooled; n=8, biologically independent animals per group. Biologically independent animals per group are shown. P-values were calculated using the two-sided Mantel-Cox test. (f) Survival rate of BALB/c mice (“secondary recipients”) which received C57BL/6 BM (5×10⁶), Ba/F3-ITD cells (day 0) and on day 2 H-2kb⁺ CD3⁺ CD8⁺ T-cells from the spleens of BALB/c mice (“primary recipients” day 12 after their BMT) which had received C57BL/6 BM (5×10⁶), T-cells (2×10⁵), and Ba/F3-ITD cells and had been treated with either vehicle or sorafenib/isotype IgG or sorafenib/anti-IL-15 antibody. The experiment was repeated three times and the results were pooled; n=13, biologically independent animals per group. Biologically independent animals per group are shown. P-values were calculated using the two-sided Mantel-Cox test. (g) The graph is showing GFP⁺Ba/F3-ITD cells (as percentages of all leucocytes) measured by flow cytometry in the blood of the different groups as described in (f) at different time points following transplantation. The experiment was repeated twice and the results (mean ± s.e.m.) were pooled, n=5, biologically independent samples per group. (h) A representative flow cytometry plot showing the percentage of GFP⁺Ba/F3-ITD cells (of all leucocytes) in the spleen on day 8 following transplantation of GFP⁺Ba/F3-ITD cells from different groups as described in (f). (i) Scatter plot showing the quantification of Bcl-2 expression levels (the fold change of MFI with respect to mean MFI of vehicle treated group) in all living donor-derived (H-2kb⁺) CD8⁺ T-cells from BMT recipients with Ba/F3-ITD cells and being treated with vehicle (n=18, biologically independent samples) or sorafenib (n=15, biologically independent samples) or from untreated naive C57BL/6 mice (n=6, biologically independent samples) as indicated. The experiment was repeated three times and the results (mean ± s.e.m.) were pooled. The P-values were calculated using the two-sided Mann-Whitney U test. (j) Scatter plot showing the quantification for PD-1 expression (the fold change of MFI with respect to mean MFI of vehicle treated group) of all living donor-derived (H-2kb⁺) CD8⁺ T-cells from BMT recipients treated with vehicle (n=18, biologically independent samples) or sorafenib (n=19, biologically independent samples) or from untreated naive C57BL/6 mice (n=6, biologically independent samples) as indicated. Each data point represents an individual sample of one biologically independent animal. The experiment was performed three times and the results (mean ± s.e.m.) were pooled. The P-values were calculated using a two-sided Student’s unpaired t-test.

Figure 4. Sorafenib induces phosphorylation of IRF7 by reducing its inhibitor ATF4

(a, b) Quantification of ATF4 (western blot) normalized to β-actin (fold change with respect to DMSO treated controls) in mouse Ba/F3-ITD (a) or human FLT3-ITD mutant MV4-11 (b) leukemia cells exposed to sorafenib as indicated. The experiment was done three times and the results (mean ± s.e.m.) were pooled; a: n=6 biologically independent samples per group, b: n=4 biologically independent samples per group. The P-values were calculated using a two-sided Mann-Whitney U test. (c) Western blots showing the expression of pIRF7, tIRF7 and loading control (β-actin) in Ba/F3-ITD cells exposed to sorafenib. Blot images were cropped and the pieces are separated by a white border. The uncut gels of all western blots are shown in Suppl. Fig.15–21. (d) Quantification of pIRF7/tIRF7 normalized to β-actin (fold change with respect to DMSO treated controls) in Ba/F3-ITD cells treated as described. The experiment was repeated four times and the results (mean ± s.e.m.) were pooled, n=5 biologically independent samples per group. Each data point represents an individual sample of one independent cell culture experiment. The P-values were calculated using a two-sided Mann-Whitney U test. (e) A representative Western blot showing the expression of pIRF7, tIRF7 and loading control (β-actin) in MV4-11 cells treated with the indicated sorafenib concentrations for 24 hours. (f) Fold change of IL-15 (MFI) in Ba/F3-ITD cells or Ba/F3-ITD cells transfected with a lentiviral vector overexpressing mouse ATF4 and when indicated treated with sorafenib (0.1 μM) for 24 hours. n=9 biologically independent cell culture samples. Each data point represents an individual sample of one independent cell culture experiment. The P-values were calculated by using a two-sided Mann-Whitney U test. (g) Percentage survival of BALB/c recipients receiving C57BL/6 BM and ATF4-overexpressing or ATF4-wildtype Ba/F3-ITD cells (500 cells) with additional C57BL/6 Tc (Tc, 2×10⁵ cells, given on day 2) and treated with vehicle or sorafenib. The experiment was performed twice and the results were pooled; n=10, biologically independent animals per group. P-values were calculated using the two-sided Mantel-Cox test. (h) Quantification of IL-15 mRNA by qPCR in MOLM-13 (human FLT3-ITD⁺ AML cell line) cells containing a non-silencing vector (MOLM-13^{NS shRNA}) or an IRF7 knockdown vector (MOLM-13^{IRF7 shRNA}). The MOLM-13 cells were exposed to the indicated concentrations of sorafenib. The experiment was repeated twice and the results (mean ± s.e.m.) were pooled, n=6 biologically independent samples per group. The P-values were calculated using a two-sided Mann-Whitney U test. (i) Percentage survival of Rag2^−/−Il2rγ^−/− recipient mice receiving MOLM-13^{NS shRNA} and MOLM-13^{IRF7 shRNA} as indicated. The experiment was performed twice and the results were pooled, n=12 biologically independent animals per group. P-values were calculated using a two-sided Mantel-Cox test. (j) Proposed mechanism as to how sorafenib leads to increased IL-15 transcription. Sorafenib inhibits FLT3 receptor tyrosine kinase signaling which normally leads to ATF4 production. Reduced ATF4 levels lead to less inhibition of IRF7 phosphorylation and activation. Active pIRF7 can translocate to the nucleus where it activates IL-15 transcription.

Figure 5. Treatment with sorafenib induces IL-15 in human primary FLT3-ITD⁺ leukemia cells

(a) Representative IL-15 mRNA levels determined by qPCR within primary human AML FLT3-ITD⁺ cells. Six technical repeats from two independent patients per group are shown. Since these results display only representative data from technical replicates no statistical analysis was performed. (b) Cumulative IL-15 mRNA levels determined by qPCR within primary human AML FLT3-ITD⁺ cells (n=8, biologically independent patients) are displayed. Each data point indicates the IL-15/GAPDH mRNA ratio in the AML cells exposed to sorafenib or DMSO. P-values were calculated using the two-sided Wilcoxon matched-pairs signed rank test and are indicated in the graph. (c) Representative IL-15 mRNA levels determined by qPCR within primary human AML FLT3-ITD^negative cells. Six technical repeats from two independent patients per group are shown. Since these results display only representative data from technical replicates no statistical analysis was performed. (d) Cumulative IL-15 mRNA levels determined by qPCR within primary human AML FLT3-ITD^negative cells (n=10, biologically independent patients) are displayed. Each data point indicates the IL-15/GAPDH mRNA ratio in the AML cells exposed to sorafenib or DMSO. P-values were calculated using the two-sided Wilcoxon matched-pairs signed rank test and are indicated in the graph.

Figure 6. Treatment with sorafenib increases the frequency of T-cells that have active glycolysis in patients with FLT3-ITD⁺ AML which relapse after allo-HCT

(a) IL-15 levels in the serum of patients relapsing with FLT3-ITD⁺ AML after allo-HCT. Sorafenib/DLI responders (n=19 biologically independent patients) and non-responders (n=14, biologically independent patients) are shown separately. Each data point indicates the IL-15 level in the serum of a patient before sorafenib (So) treatment (day 0) and after start of sorafenib-treatment (day 3 after start of treatment and before the patients received donor lymphocyte infusions, DLI). The dotted line in the graph indicates the detection limit (4 pg/ml) of the IL-15 ELISA. The P-value was determined using the two-sided Wilcoxon matched-pairs signed rank test and is indicated in the graph. (b) Quantification of IL-15 mRNA expression by qPCR in leukemia cells (>90% purity) derived from the peripheral blood of FLT3-ITD⁺ AML patients (responders n=14 biologically independent patients, non-responders, n=14 biologically independent patients) before (day -3) and day 6 after start of sorafenib-treatment. Each data point indicates the measurement of an individual patient at the indicated time point. The P-value was determined by using the two-sided Wilcoxon matched-pairs signed rank test. (c) Quantification of pIRF7⁺ cells/HPF in BM biopsies of FLT3-ITD⁺ AML patients (responders n=12 biologically independent patients, non-responders, n=12 biologically independent patients) before (day 0) and during sorafenib-treatment (day 15 after start of treatment). Each data point indicates the measurement of an individual patient at the indicated time point. The P-value was determined by using the two-sided Wilcoxon matched-pairs signed rank test. (d) IFN-γ levels in the serum of patients relapsing with FLT3-ITD⁺ AML after allo-HCT. Sorafenib/DLI responders (n=19, biologically independent patients) and non-responders (n=14, biologically independent patients) are shown separately. Each data point indicates the IFN-γ level in the serum of a patient before (day 0) and after start of sorafenib-treatment (day 3 after start of treatment and before the patients received donor lymphocyte infusions, DLI). Responders: IFN-γ (serum level) before versus after sorafenib, P=0.007. Non-responders: no significant difference before vs after sorafenib. The P-value was determined using the two-sided Wilcoxon matched-pairs signed rank test. (e) The heatmap displays the significance in having a smaller number of germline and somatic mutations in chromatin states marked as Tx (strong transcription) or TxW (weak transcription) around the transcription start site (TSS) of various interferon genes for non-responders (n=4, biologically independent patients) versus responders (n=4, biologically independent patients). IRF genes were hierarchically clustered by their Euclidean distance using complete linkage algorithm. Significance in mutation frequency was calculated from an analysis of variance with posthoc Tukey’s test. P-values for the comparison of the different IRFs for responders vs non-responders were as follows; IRF7 P=0.0005, IRF5: P=0.001 (like IRF7 the IRF5 activation induces IFN-responses ), not significant for: IRF1-IRF4, IRF6-IRF9. (f) ECAR rates of CD8⁺ T-cells after FCCP exposure, derived from the peripheral blood of FLT3-ITD⁺ AML patients (only responders n=12, biologically independent patients) before (day-2) and during sorafenib-treatment (day 4 after start of treatment). Each data point indicates the measurement of an individual patient at the indicated time point. The P-value was determined by using the two-sided Wilcoxon matched-pairs signed rank test. (g) Change in OCR relative to basal level after FCCP exposure of CD8⁺ T-cells, derived from the peripheral blood of FLT3-ITD⁺ AML patients (only responders n=12, biologically independent patients) before (day-2) and during sorafenib-treatment (day 4 after start of treatment). Each data point indicates the measurement of an individual patient at the indicated time point. The P-value was determined by using the two-sided Wilcoxon matched-pairs signed rank test. Abbreviations: extracellular acidification rate (ECAR; reflecting the rate of glycolysis indicated by lactate secretion) and oxygen consumption rate (OCR; reflecting OXPHOS). (h) ECAR rates of CD8⁺ T-cells after FCCP exposure, derived from the peripheral blood of FLT3-ITD⁺ AML patients (only non-responders n=14, biologically independent patients) before (day-2) and during sorafenib-treatment (day 4 after start of treatment). Each data point indicates the measurement of an individual patient at the indicated time point. The P-value was determined by using the two-sided Wilcoxon matched-pairs signed rank test. (i) Change in OCR relative to basal level after FCCP exposure of CD8⁺ T-cells, derived from the peripheral blood of FLT3-ITD⁺ AML patients (only non-responders n=14, biologically independent patients) before (day-2) and during sorafenib-treatment (day 4 after start of treatment). Each data point indicates the measurement of an individual patient at the indicated time point. The P-value was determined by using the two-sided Wilcoxon matched-pairs signed rank test. (j) Comparison of the median diversity index (DI) of TCR α and β complementarity determining region 3 (CDR3) amino acid sequences between responders and non-responders is shown. The analysis was performed on CD3⁺ cells isolated from responders (n=7, biologically independent patients) and non-responders (n=7, biologically independent patients) on day 30 after start of sorafenib. The P-value was determined by using the two-sided Mann-Whitney U test. No adjustments were made for multiple comparisons.