Fig 1: 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 2: Analysis of the RIT1-RAF RBD structural interface.(A) Comparison of modeled structures of WT and A57G mutant of RIT1 with the crystal structure of KRAS:RAF1(RBD) complex (PDB: 6VJJ). Top left: Interaction formed by KRAS Ser39 (equivalent to Ala57 in RIT1) and neighboring residues Asp38 and Tyr40 with RAF1-RBD. KRAS and RAF1-RBD are colored green and cyan, respectively. Top right: Superposition of KRAS:RAF1(RBD) complex with the modeled structure of WT RIT1 (colored yellow) in the active state. Bottom left: Superposition of KRAS:RAF1(RBD) complex with the modeled structure of A57G mutant of RIT1 (colored pink) in the active state. Bottom right: Superposition of KRAS:RAF1(RBD) complex with the modeled structures of WT and A57G mutant of RIT1 in the active state. (B and C) Immunoblot analysis of indicated proteins immunoprecipitated from HEK293T cell lysates expressing indicated constructs. (D) Immunoblot analysis of indicated proteins from HEK293T cells transiently transfected with indicated FLAG-tagged RIT1 constructs or an EV. For (B) to (D), one of two independent experiments is shown.
Fig 3: 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.
Fig 4: Knockdown of NECAB3 suppressed the activation of the HIF-1α/RIT1 pathway. The protein levels of HIF-1α and RIT1 in Li7 and HCCLM3 cells transfected with siNECAB3 and overexpression vector of NECAB3 were analyzed by Western blot. ***: P < 0.001. ##: P < 0.01.
Fig 5: RIT1 oncoproteins activate ERK signaling via RAF kinase.(A) Immunoblot analysis of indicated proteins from HEK293T cells transiently transfected with indicated FLAG-tagged RIT1 constructs or an EV control and treated with 10 μM LY3009120 (RAFi) or DMSO vehicle control for 1 hour. One of two independent experiments is shown. (B) Immunoblot analysis of indicated proteins from HEK293T cells transiently transfected with indicated siRNA and FLAG-tagged RIT1 constructs or EV control. One of two independent experiments is shown. (C) In vitro MEK1 phosphorylation (Ser218/Ser222) by RAF1 or BRAF protein isolated from HEK293T cells coexpressing RIT1A57G, KRASQ61L, or an EV control. a.u., arbitrary units. Data points indicate the means ± SEM of four (RAF1) or three (BRAF) biological replicates (independently isolated RAF protein samples). p, phospho.
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