Journal: Cancer discovery

Article Title: Impaired proteolysis of non-canonical RAS proteins drives clonal hematopoietic transformation

doi: 10.1158/2159-8290.CD-21-1631

Figure Lengend Snippet: (A) Lollipop plot of LZTR1 mutations identified in blood of subjects with clonal hematopoiesis (7). (B) Representative histograms of GFP in 293T cells encoding RIT1 fused to eGFP and empty vector (Vector), wild-type (WT) LZTR1, or any of four CH-associated LZTR1 mutations. % of eGFP+ cells 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 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 wild-type or null embryos for cleaved caspase 3 (green), Ter119 (red), and DAPI (blue) in whole mount (top; WT bar: 100μm; KO bar: 50μm) or focused on fetal livers (bottom; bar, 20μm). (G) Schema of experiments evaluating 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) % of CD45.2+ cells in peripheral blood of CD45.1+ recipient mice following primary and secondary 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. n=5. **p<0.01. (K) % 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. (L) Kaplan-Meier curve of primary and secondary transplant recipient mice. Pie chart indicates 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. (N) Flow cytometric analysis of live, CD45.2+ Lztr1−/− cells from secondary transplant recipient mice developing myeloid neoplasms or B-cell acute lymphoblastic leukemias.

Article Snippet: RIT1 degradation reporter assay RIT1 and RIT1 M90I mutant cDNAs were cloned into an enhanced green fluorescent protein (eGFP) degradation reporter (Addgene #74459).

Techniques: Plasmid Preparation, Disruption, Western Blot, Knock-Out, Immunofluorescence, In Vitro, In Vivo, Transplantation Assay, Whisker Assay

Journal: Cancer discovery

Article Title: Impaired proteolysis of non-canonical RAS proteins drives clonal hematopoietic transformation

doi: 10.1158/2159-8290.CD-21-1631

Figure Lengend Snippet: (A) Histogram of number of patients with RIT1 mutations based on myeloid malignancy diagnosis. Abbreviations: AML: acute myeloid leukemia; MDS: myelodysplastic syndromes; MDS/MPN: myelodysplastic syndromes/myeloproliferative neoplasms; MF: myelofibrosis; No Dx: no diagnosis. (B) Diagram of location of RIT1 mutations identified. (C) Variant allele frequency (VAF) of mutations in RAS GTPases or regulators of RAS GTP abundance relative to mutations in transcriptional modifiers in myeloid leukemia patients. (D) Fish tail representation plots of VAFs of mutations across serial genomic analysis of three RIT1 mutant patients. (E) Western blot of LZTR1 and RIT1 following cycloheximide (CHX) treatment of TF-1 cells with or without LZTR1 deletion. (F) Representative histograms of GFP in cells encoding wild-type or mutant RIT1 fused to eGFP along with empty vector (EV), wild-type LZTR1, or mutant LZTR1. % of eGFP+ cells indicated. Red dotted line indicates the cutoff for GFP+. (G) Levels of phosphorylated and total MEK1/2 and ERK1/2 as well as RIT1 in TF-1 cells with LZTR1 deletion or expression of empty vector RIT1 wild-type (WT) or mutant cDNAs. (H) Western blot of pERK and total ERK levels in 293T cells transfected with increasing amounts of FLAG-RIT1 wild-type (WT) and mutant cDNAs.

Article Snippet: RIT1 degradation reporter assay RIT1 and RIT1 M90I mutant cDNAs were cloned into an enhanced green fluorescent protein (eGFP) degradation reporter (Addgene #74459).

Techniques: Biomarker Discovery, Variant Assay, Mutagenesis, Western Blot, Plasmid Preparation, Expressing, Transfection