Fig 2: YTHDF2 Is Upregulated in Different AML Subtypes and Is Essential for AML Development(A) YTHDF2 gene expression in control (CTL) and different cytogenetic subgroups of human AML bone marrow samples. Violin plots show the distribution of log2 expression values. Horizontal line in the boxplots indicates median. CNG, cytologically normal with good prognosis; CNI, cytologically normal with intermediate prognosis; CAO, cytologically abnormal not otherwise specified.(B) Western blot of YTHDF2 in normal human CD34+ cells and AML samples (karyotype details are shown in STAR Methods) (left). α-Histone 3 (H3) was used as a loading control. Quantification of YTHDF2 normalized to H3 expression is presented (right).(C) YTHDF2 gene expression in primitive AML cell compartments with (LSC+) and without (LSC−) leukemic engraftment potential.(D) Control Ythdf2fl/fl (Ythdf2CTL) and Ythdf2fl/fl;Vav-iCre (Ythdf2CKO) fetal liver (FL) c-Kit+ cells were co-transduced with Meis1 and Hoxa9 retroviruses and serially replated. c-Kit+ preleukemic cells were transplanted into recipient mice (n = 12–14).(E) A representative histogram showing GFP-YTHDF2 protein expression in Ythdf2CTL FL LSK cells and the lack of GFP-YTHDF2 expression in Ythdf2CKO FL LSK cells.(F) Percentage of GFP-positive cells in the 14.5 days post coitum (dpc) FL LSK cell population from FLs of Ythdf2CTL and Ythdf2CKO embryos (n = 5).(G) CFC counts at each replating (n = 3).(H) Percentage of CD11b+Gr-1−, CD11b+Gr-1+, and c-Kit+ cells in the preleukemic cell compartment (n = 4–5).(I) Percentage of CD45.2+ leukemic cells in the PB of recipient mice (n = 12–14 per genotype).(J) Survival curve of recipients transplanted with preleukemic cells (n = 12–14).(K) Percentage of GFP-positive cells in the CD45.2+ cell population from moribund recipients of Ythdf2CTL and Ythdf2CKO cells (n = 5–6).(L) Limiting dilution assay (LDA). Secondary recipients (n = 5–8) were transplanted with indicated doses of CD45.2+ BM cells from primary recipients.(M) Ythdf2CTL and Ythdf2CKO FL c-Kit+ cells were transduced with MOZ-TIF2 or PML-RARA retroviruses and serially replated. CFC counts at each replating are shown (n = 3).Data represent mean ± SEM; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001.

Fig 3: Conventional dendritic cells accumulate in the dural meninges during chronic brain infection and sample CSF-derived protein.Mice were infected with 10 cysts of the ME49 strain of T. gondii intraperitoneally (i.p.) and analyzed at 6 weeks post-infection. (a) Confocal microscopy was used to image whole-mount dural meninges dissected from the skullcaps of naïve or chronically infected CD11cYFP reporter mice. Images are representative of three independent experiments. Scale bar, 2000 µm. (b) Representative images of CD11c+ cells (yellow) in the region surrounding the dural sinuses. Scale bar, 150 µm. (c) Quantification of area coverage by CD11c+ cells in the region surrounding the dural sinuses. Data compiled from three experiments (n = 10-11 mice per group). (d-e) Representative images (d) and quantification (e) of CD11c+ cells (yellow) present within LYVE1+ meningeal lymphatic vessels (white). Data compiled from three experiments (n = 10-11 mice per group). Scale bar, 50 µm. (f) Quantification by spectral flow cytometry of total cDC1 number (CD45+Lin-CD11chiMHC IIhiCD64-CD26+XCR1+SIRPa-) in the dural meninges of naïve, acutely infected, or chronically infected C57BL/6 mice. Data compiled from two experiments (n = 7 mice per group). (g) Quantification by spectral flow cytometry of total cDC2 number (CD45+Lin-CD11chiMHC IIhiCD64-CD26+XCR1-SIRPa+) in the dural meninges of naive, acutely infected, or chronically infected C57BL/6 mice. Data compiled from two experiments (n = 7 mice per group). (h) Quantification of T. gondii gDNA in brain and dural meninges by real-time PCR. Data compiled from two experiments (n = 3-8 mice per group). (i–k) Quantification of the geometric mean fluorescence intensity (MFI) of CD80 (i), CD86 (j), and MHC class II (k) expressed by cDC1s and cDC2s in the dural meninges. Data compiled from two experiments (n = 7 mice per group). (l) Schematic diagram illustrating intra-cisterna magna (i.c.m.) injection of DQ-OVA into the CSF. (m) Representative images of MHC class II-expressing antigen-presenting cells (magenta) co-labeling with fluorescent DQ-OVA cleavage products (green) in the vicinity of (closed arrows) or present within (open arrows) meningeal lymphatic vessels (LYVE1+, white). Three independent experiments were performed. Scale bar, 50 µm. Data are represented as mean values ± s.e.m. For c and e, statistical significance was measured using randomized block ANOVA (two-way), with p<0.001 (***). For (f and g) statistical significance was measured using a one-way ANOVA with post-hoc Tukey multiple comparison testing, with p<0.05 (*), p<0.01 (**), and p<0.001 (***). For (h–k) statistical significance was measured using a two-way ANOVA, with Tukey’s multiple comparison test to assess differences across timepoints [p<0.01 (##) and p<0.001 (###)] and Sidak’s multiple comparison test to assess differences between tissues (h) or cell type (i–k) [ns = not significant, p<0.05 (*), p<0.01 (**), and p<0.001 (***)].

Fig 4: Loss of Lztr1 enhances HSC self-renewal and drives leukemia development. A, Lollipop plot of LZTR1 mutations identified in the blood of subjects with clonal hematopoiesis (7). B, Representative histograms of GFP in 293T cells encoding RIT1 fused to eGFP and empty vector (EV), WT LZTR1, or any of four CH-associated LZTR1 mutations. The percentage of eGFP+ cells is indicated. Red dotted line indicates the cutoff for GFP+. C, Quantification of data from B. Mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. D, Schema of the allele for Lztr1 constitutive or conditional gene disruption. E, Western blots of Lztr1 and RAS GTPases in E14.5 fetal liver cells from Lztr1 WT or knockout embryos. F, Immunofluorescence images of Lztr1 WT or null embryos for cleaved caspase-3 (green), Ter119 (red), and DAPI (blue) in whole mount (top; WT bar: 100 μm; KO bar: 50 μm) or focused on fetal livers (bottom; bar, 20 μm). G, Schema of experiments evaluating the effects of Lztr1 deletion on fetal hematopoietic cells in vitro and in vivo. H, Colony number in methylcellulose replating assays using 20 × 103 fetal liver hematopoietic cells from Lztr1+/+, Lztr1+/−, and Lztr1−/− fetus. Mean ± SD. n = 3. ***, P < 0.001; ****, P < 0.0001. I, The percentage of CD45.2+ cells in the peripheral blood (PB) of CD45.1+ recipient mice following primary (1°) and secondary (2°) competitive transplantation. n = 5–10. **, P < 0.01; ****, P < 0.0001. J, Box-and-whisker plots of the percentage of total CD45.2+ cells in the bone marrow (BM). n = 5. **, P < 0.01. K, The percentage of CD45.2+ hematopoietic stem and progenitors following 16 weeks of competitive transplantation (as shown in the schema in Fig. 1G). n = 5. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. CMP, common myeloid progenitor; GMP, granulocyte–macrophage progenitor; LT-HSC, long-term HSC; LSK, lineage-negative Sca-1+ c-Kit+; MEP, megakaryocyte–erythroid progenitor; MPP, multipotent progenitor; ST, short-term HSC. L, Kaplan–Meier curve of primary and secondary transplant recipient mice. Pie chart indicates the number and proportion of analyzed mice developing lethal hematopoietic malignancies across both primary and secondary transplantation. Lztr1+/+ primary (1o) recipient mice n = 7, Lztr1−/− primary (1o) recipient mice n = 7, Lztr1−/− secondary (2o) recipient mice n = 30. *, P < 0.05; ****, P < 0.0001. M, Peripheral blood counts of CD45.1+ recipient mice transplanted with CD45.2+Lztr1+/+ or Lztr1−/− fetal liver cells. n = 6–10. **, P < 0.01; ****, P < 0.0001. HGB, hemoglobin; WBC, white blood cell. N, Flow-cytometric analysis of live CD45.2+Lztr1−/− cells from secondary transplant recipient mice developing myeloid neoplasms or B-ALL.

Fig 5: Rit1 M90I/WT mutation drives the development of myeloid neoplasms in vivo. A, Kaplan–Meier curve of primary Mx1-cre Rit1M90I/WT mice following polyinosinic:polycytidylic acid (pIpC) treatment. Venn diagram indicates diagnoses of Rit1M90/+ mice at the time of death and proportion developing MPN and myelodysplasia/MPN (MDS/MPN). n = 12. ***, P < 0.001. Wright–Giemsa stain of peripheral blood (B) and bone marrow (C) cytospins of Mx1-cre control and Mx1-cre Rit1M90I/WT mice at the time of disease onset of the Rit1M90I/WT mice. Red arrows indicate dysplastic erythroid precursors. Green arrows indicate dysplastic neutrophils. Yellow arrow indicates dysplastic myeloid precursors. Two representative animals are shown for the Rit1M90I/WT mice. D, Hematoxylin–eosin stain of bone marrow (yellow arrows indicate dysplastic megakaryocytes; scale bars = 100 μm). Two representative animals are shown for the Rit1M90I/WT mice. E, Box-and-whisker plots of peripheral blood counts of primary Mx1-cre Rit1M90I/WT mice and their age-matched Mx1-cre control mice from A. For box-and-whisker plots, bar indicates median; box edges, first and third quartile values; and whisker edges, minimum and maximum values. n = 12. *, P < 0.05; **, P < 0.01; ***, P < 0.001. WBC, white blood cell. F, The percentage of CD45.2+ cells in peripheral blood of sublethally irradiated CD45.1+ recipient mice following transplantation of Mx1-cre Rit1M90/WT bone marrow cells from the time of disease onset from D. n = 5–10. Mean ± SD. ****, P < 0.001. G, Kaplan–Meier curve of CD45.1 recipient mice from F. n = 5–10. ***, P < 0.001.