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

Journal: Science (New York, N.Y.)

Article Title: Defining the human C2H2 zinc-finger degrome targeted by thalidomide analogs through CRBN

doi: 10.1126/science.aat0572

Figure Lengend Snippet: (A) TR-FRET counter titration. Unlabeled IKZF1 constructs (0.01–100 μM) titrated to preassembled Alexa488DDB1ΔB-CRBN-pomalidomide-biotinIKZF1ZF1−2−3 (200 nM Alexa488DDB1ΔB-CRBN, 100nM biotinIKZF1ZF1−2−3, 5μM pomalidomide). (B) Schematic of the protein degradation reporter vector (IRES: Internal ribosome entry site). (C) HEK293T WT and CRBN−/− cells expressing IKZF3 constructs in the degradation reporter were treated for 20 hours with DMSO or drug then analyzed by flow cytometry to quantify the DMSO-normalized ratio of eGFP/mCherry fluorescence (exp. rep. = 3, tech. rep. = 3, bar heights indicate mean of experimental replicates, error bars indicate 95% CI). (D) Schematic of the human C2H2 ZF library screen. (E) Average fold-depletion of sequencing read counts (DMSO/drug) and corresponding p values (empirical rank-sum test-statistic) for the 5,611 C2H2 ZFs with raw read count >200 in all three control replicates (exp. rep. = 3, labeled data points possess FDR<0.01 in at least one of the three drugs).

Article Snippet: 1 , Artichoke , SFFV.BsmBICloneSite- 17aaRigidLinker -EGFP.IRES.mCherry.cppt.EF1α.PuroR , AmpicillinR , Lentiviral (pLKO, taken from lentiGuide-Puro, Addgene #52963) , #73320 , Full-length proteins, IKZF3 aa130–189, saturation mutagenesis screen.

Techniques: Titration, Construct, Plasmid Preparation, Expressing, Flow Cytometry, Fluorescence, Sequencing, Control, Labeling

Journal: Science (New York, N.Y.)

Article Title: Defining the human C2H2 zinc-finger degrome targeted by thalidomide analogs through CRBN

doi: 10.1126/science.aat0572

Figure Lengend Snippet: (A) Sequence alignment of the 11 C2H2 ZFs with FDR<0.01 in at least one drug condition (amino acids colored by property). (B) Saturation mutagenesis screen of IKZF3 aa 130–189 in presence of lenalidomide displayed as heat map of the FDR for mutant amino acids (unpaired, one-sided t-test, FDR correction performed within each column, tech. rep. = 3). Asterisks indicate amino acids required for the ZF fold and arrows indicate non-structural IKZF3 residues required for degradation. Complete results for all 60 amino acids are located in fig. S3C (C) ANOVA p values for difference in frequency of mutant amino acids at each position in IKZF3 aa 130–189 (DMSO vs drug). Complete results for all 60 aa are located in fig. S3D. (D) HEK293T cells expressing IKZF3 ZF2 constructs in the degradation reporter were treated for 20 hours with DMSO or 1μM drug after which flow cytometry was used to measure the DMSO-normalized ratio of eGFP/mCherry fluorescence (exp. rep. = 1, tech. rep. = 3, bar height is the average of tech. rep, error bars denote 95% CI).

Article Snippet: 1 , Artichoke , SFFV.BsmBICloneSite- 17aaRigidLinker -EGFP.IRES.mCherry.cppt.EF1α.PuroR , AmpicillinR , Lentiviral (pLKO, taken from lentiGuide-Puro, Addgene #52963) , #73320 , Full-length proteins, IKZF3 aa130–189, saturation mutagenesis screen.

Techniques: Sequencing, Mutagenesis, Expressing, Construct, Flow Cytometry, Fluorescence

Journal: Science (New York, N.Y.)

Article Title: Defining the human C2H2 zinc-finger degrome targeted by thalidomide analogs through CRBN

doi: 10.1126/science.aat0572

Figure Lengend Snippet: (A) Side chain interactions between IKZF1 ZF2, CRBN, and pomalidomide. Dashed lines indicate hydrogen bonds. (B) Chemical structures of pomalidomide, lenalidomide, and thalidomide with differences highlighted in red. (C) Inhibitory constants (Ki) for wild-type (WT) and Q146I mutant IKZF1ZF2-ZF3 in the presence of thalidomide, lenalidomide, and pomalidomide in TR-FRET counter titration experiments. (D) HEK293T cells expressing IKZF3 ZF2 Q147 mutants in the degradation reporter were treated for 20 hours with DMSO or drug and then analyzed by flow cytometry to measure the DMSO-normalized ratio of eGFP/mCherry fluorescence (exp. rep. = 3, tech. rep. = 3, bar height is average of exp. rep, error bars indicate 95% CI). The complete set of Q147 amino acid mutants is located in fig. S5D.

Article Snippet: 1 , Artichoke , SFFV.BsmBICloneSite- 17aaRigidLinker -EGFP.IRES.mCherry.cppt.EF1α.PuroR , AmpicillinR , Lentiviral (pLKO, taken from lentiGuide-Puro, Addgene #52963) , #73320 , Full-length proteins, IKZF3 aa130–189, saturation mutagenesis screen.

Techniques: Mutagenesis, Titration, Expressing, Flow Cytometry, Fluorescence

Journal: Science (New York, N.Y.)

Article Title: Defining the human C2H2 zinc-finger degrome targeted by thalidomide analogs through CRBN

doi: 10.1126/science.aat0572

Figure Lengend Snippet: Degradation Reporter Vectors

Article Snippet: 1 , Artichoke , SFFV.BsmBICloneSite- 17aaRigidLinker -EGFP.IRES.mCherry.cppt.EF1α.PuroR , AmpicillinR , Lentiviral (pLKO, taken from lentiGuide-Puro, Addgene #52963) , #73320 , Full-length proteins, IKZF3 aa130–189, saturation mutagenesis screen.

Techniques: Marker, Mutagenesis, Zinc-Fingers, Isolation