A broad range of second-site TpoR TMD mutations enhance the cytokine-independent activity of the S505N mutant receptor. (A) Sequence-function map showing the results of saturation mutagenesis of human TpoR-S505N TMD (residues 488-516). Color of each square indicates the log ratio enrichment score for variant frequencies in Ba/F3 cells cultured for 48 hours without cytokines compared with the unselected library, according to the scale below the map. Scores reflect variant frequency changes relative to S505N (white squares with dots; score of 0). Red shades indicate enrichment. Blue shades indicate depletion. Diagonal lines are proportional to standard deviations of scores across 6 replicate selections. (B) A selection of double mutants from (A) were tested in a competition assay in which TpoR-S505N (reference) cells expressed mCherry and double mutants (challengers) expressed green fluorescent protein (GFP). Approximately equal numbers of cells (GFP-to-mCherry ratio ∼1) were coseeded in the same well and grown for 96 hours without cytokines. Relative levels of GFP⁺ and mCherry⁺ cells were determined daily by flow cytometry and are plotted as the GFP-to-mCherry ratio. Data points represent the mean of 3 technical replicates from 1 of 3 independent experiments performed. Error bars show standard deviations. Data for additional experiments are shown in supplemental Figure 3. Doubling times (mean and standard deviation) were calculated using combined data from all 3 independent experiments. Significance values are given for each challenger’s doubling time compared with that of S505N (1-way ANOVA: ****P < .0001). Doubling times >96 hours indicate rapid death or no measurable growth. (C) Competition assay performed as in panel B testing the patient-derived TpoR H499G+V501S²⁷  double mutant (mCherry) coseeded with GFP-expressing WT (left) or V501S (right). Plots show percentage of GFP and mCherry cells in the mixed cultures over 96 hours of growth. Data points represent the mean of 3 independent experiments. Error bars are standard deviations. (D) MPN patients identified with multiple MPL exon 10 mutations. (E) One variant allele frequency (VAF) value for this patient reflects the frequency of the paired mutations. *Patient with triple MPL exon 10 mutation was also JAK2-V617F⁺ (VAF 100%). All other patients were WT at JAK2 and CALR exon 9. Where VAF values, knowledge of familial disease, and/or availability of lymphocyte DNA sequences allow differentiation between germline (Ger) and somatic (Som) lesions, individual mutations are marked in superscript. F, female; M, male; MF, myelofibrosis; ND, not determined; PLT, platelet count at diagnosis (×10⁶/mL).