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. 2022 Jan 1;107(1):342-346.
doi: 10.3324/haematol.2021.279601.

Tumor suppressor function of WT1 in acute promyelocytic leukemia

Matthew J Christopher  1 , Casey D S Katerndahl  2 , Hayley R LeBlanc  2 , Tyler T Elmendorf  2 , Vaishali Basu  2 , Margery Gang  2 , Andrew J Menssen  2 , David H Spencer  2 , Eric J Duncavage  3 , Shamika Ketkar  4 , Lukas D Wartman  2 , Sai Mukund Ramakrishnan  2 , Christopher A Miller  2 , Timothy J Ley  2
Affiliations

Tumor suppressor function of WT1 in acute promyelocytic leukemia

Matthew J Christopher et al. Haematologica. .
No abstract available

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Figures

Figure 1.
Figure 1.
WT1 expression is induced in human CD34+ cells transduced with RUNX1-RUNX1T1, PML-RARA, or MYC. Umbilical cord blood-derived CD34+ cells were cultured in cytokines after transduction with GFP-tagged retroviruses expressing RUNX1-RUNX1T1, MYC, PML-RARA, or an empty vector control. (A) WT1 expression is induced in CD34+ cells transduced with RUNX1-RUNX1T1, MYC, or PML-RARA compared to controls transduced with empty vector (GFP, green) or untransduced cells (CD34, pink). CD34+ cells transduced with each vector (n=2-6 separate experiments) were cultured for 7 days, RNA was isolated from flow-sorted GFP+ cells, and WT1 mRNA was quantified by real time polymerase chain reaction. P-values were calculated using Student’s t-test. (B) Western blot showing expression of WT1 in CD34+ cord blood cells 7 days after transduction with RUNX1-RUNX1T1 (AE), PML-RARA (PR), MYC, empty vector (GFP), or untransduced (CTRL). Lysates were made from sorted GFP+ cells except control (CTRL), which was made from equivalent cell numbers of untransduced cells cultured in parallel. Blot represents one of 3 representative experiments. (C) Heatmaps showing differentially expressed genes (DEG) in human or mouse cells transduced with a PML-RARA-expressing MSCV vector. Human CD34+ cells or mouse lineage-depleted bone marrow cells were transduced with IRES-GFPtagged retroviruses containing a PML-RARA cDNA, or no insert (empty vector). After 7 days in culture, GFP+ cells were flow sorted and RNA was isolated for RNA sequencing. DEG were identified using a false discovery rate (FDR) cutoff of <0.05 after filtering out genes with low expression across all samples (see the Online Supplementary Appendix). Heatmaps show DEG in PML-RARA vs. empty vector-transduced human (n=2 separate experiments) and mouse (n=3 separate experiments) progenitor cells. (D) Venn diagram showing overlap in orthologous mouse and human DEG from (C). Of 4,915 mouse DEG having human orthologues, 867 are DEG in the analysis of human genes (P=9.6x10-118 using the hypergeometric test). (E) WT1 expression is increased by PML-RARA transduction in human CD34+ cells (left panel), but not in mouse bone marrow-derived cells (right panel). WT1/Wt1 expression values from the RNA sequencing experiment described above are shown. P-values were calculated using Student’s t-test. TPM: transcripts per million.
Figure 2.
Figure 2.
Inactivating mutations in WT1 provide a growth advantage for PML-RARA-transduced CD34+ cells. (A) Umbilical cord blood-derived CD34+ cells were transduced with GFP-tagged lentiviruses encoding the two most common WT1 isoforms (KTS+ and KTS-), or an empty vector. Cells were maintained in culture with cytokines, and GFP+ cells were quantified at different time points. Shown are percent GFP+ cells over time in WT1 (right) or empty vector (left) transduced cultures normalized for transduction efficiency at beginning of the culture period (n=4 individual experiments). Black dotted lines show line of best fit calculated by linear regression. Transduction with WT1 isoforms (KTS+ and KTS-) leads to loss of GFP+ cells (slope b=-1.18 per day, P<0.001), while empty vector-transduced cells have GFP+ cells throughout the culture period (slope b=-0.54, P=0.32). P-values were calculated using a linear regression model, and represent the probability that the slope of the best fit line equals zero. (B) Human CD34+ cord blood cells were transduced with PML-RARA-expressing retrovirus or empty vector, and 48 hours later CRISPR/Cas9 was used to generate mutations in WT1 (exon 1 or exon 8) or AAVS1 (a negative control locus). GFP+ cells were sorted at different time points from cultures that had been transduced with a vector containing PML-RARA (right panels) or no insert (empty vector, left panels). DNA was isolated and polymerase chain reaction products containing the guide RNA target sites were digitally sequenced to determine the precise variant allele frequencies of mutations in WT1 (bottom panels) or AAVS1 (top panels). Shown are change in variant allele frequency (VAF) of AAVS1 mutations or WT1 mutations over time (n=3-6 separate experiments). Mutations in WT1 exon1 are shown in green, or WT1 exon 8 in red. Black dotted lines show line of best fit calculated by linear regression. Cells containing mutations in WT1 show a trend toward expansion in empty vector-transduced CD34+ cells (slope b=0.003 increase per day, P=0.20), and a statistically significant expansion in PML-RARA-transduced cells (slope b=0.006 increase per day, P=0.007). In contrast, cells with mutations in AAVS1 do not expand over time. P-values were calculated using a linear regression model, and represent the probability that the slope of the best fit line equals zero. (C) Increase in overall cell numbers in cultures transduced with GFP (left) or PML-RARA (right). P-values were calculated using Student’s t-test.
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References

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