Extended Data Fig. 5: Supplemental experiments functionally validating DYRK1A as a target in BP-MPN.

a. Cell counts for two SET2 DYRK1A KO clones (14B5 & 11H1) generated by CRISPR/Cas9 and expanded in culture, confirming adverse effect on proliferation by DYRK1A KO over time (Shown are mean +/- SEM, n=3 biological replicates, p<0.05 by day 5 by ANOVA). b. Western blot showing the knockout of DYRK1A in the 14B5 & 11H1 SET2 cell clones. Densitometric values normalized to HSC70 (representative of n=3 experiments). c. Cell proliferation assay for SET2 cells transduced with lentiviruses expressing DYRK1A specific shRNA or scramble control (Shown are mean +/- SEM, n=3 biological replicates, p<0.05 by day 5, by ANOVA). d. Western blot showing the knockdown of DYRK1A expression in SET2 cells following transduction with lentiviruses expressing target specific shRNA or scramble control. Densitometric values were normalized to actin (representative of n=3 experiments). e. Independent replicate experiment of shRNA knockdown experiments. HEL cells were treated with lentiviruses expressing DYRK1A specific shRNA or scramble control, and placed in the Incucyte for serial cell counts (summary of 3 biological replicates, mean +/- SEM, p<0.001 by ANOVA). f. Knockdown of DYRK1A was validated by qRT-PCR (n=3 replicates, shown are mean +/- SEM, compared by t-test, p-values shown are two-sided). g. Western blots for phosphorylation of LIN52 at S28, FOXO1 at S329, total LIN52 and total FOXO1 protein levels after 4 hours of treatment with increasing doses of the DYRK1A inhibitor EHT1610 in SET2 cells are shown. Densitometric values were normalized to HSC70 and GRB2 protein levels, respectively (representative of n=2 experiments). h. Experimental layout for primary patient experiments. i. Timecourse of viability readouts for primary patient cells on days 1,5,8 post treatment with DYRK1A inhibitors EHT1610 and GNF2133 at 0.1 and 1μM doses. On the y axis is % cell death relative to DMSO control. Each sample (n=5 healthy controls, n=4 non-chr21amp and n=4 chr21amp patients) is shown by a dot, with mean and SD depicted in the boxplot. Significance testing by t-test with Bonferroni correction for multiple testing, shown are the adjusted q values *<0.1 **<0.05, ***<0.001.