Page 350 - 2022_01-Haematologica-web

Letters to the Editor
Tumor suppressor function of WT1 in acute promye- locytic leukemia
Originally identified as a cancer susceptibility gene, Wilms’ Tumor 1 gene (WT1) is overexpressed or mutated in a wide variety of malignancies, including acute myeloid leukemia (AML). WT1 is a zinc-finger transcrip- tion factor comprised of C-terminal zinc-finger DNA binding domains and an N-terminal transactivation domain thought to regulate interactions with partner proteins. Germline WT1 mutations consist primarily of nonsense mutations that truncate the C-terminal domains, or missense mutations that disrupt DNA bind- ing, and these mutations result in both developmental abnormalities and predisposition to Wilms’ tumor.1
In normal human CD34+ hematopoietic stem/progen- itor cells (HSPC), wild-type WT1 is expressed at a low level, but it is highly expressed in nearly all cases of AML. Among all AML subtypes, WT1 expression is gen- erally highest in acute promyelocytic leukemia (APL/M3 AML), the AML subtype initiated by the PML-RARA fusion gene (Online Supplementary Figure S1A and D).2,3 In addition, we and others4 have identified recurrent WT1 mutations in APL cases (11/42 [26%] in this study) (Online Supplementary Figure S1C). In seven of 11 of these cases, WT1 mutations occurred at a significantly lower variant allele frequency (VAF) than PML-RARA, suggest- ing they are co-operating events in subclones (data not shown). WT1 mutations have been associated with worse prognosis in non-M3 AML, although no such association has been shown in APL. The spectrum of WT1 mutations is similar in APL versus other AML cases (Online Supplementary Figure S1B), suggesting that WT1 mutations may have similar biologic activities across all AML subtypes.
These observations frame a well-known - but unsolved - paradox that we attempt to address here: does a high level of wild type WT1 expression contribute to the initiation or progression of AML/APL, or con- versely, does it reflect a tumor suppressor activity, since inactivating mutations appear to contribute to disease progression?
In order to explore these questions, we first tested the ability of Wt1 mutations to co-operate with PML-RARA in a well-characterized murine APL model. Ctsg-PML- RARA mice express the PML-RARA fusion cDNA in immature hematopoietic progenitor cells, and succumb to an APL-like disease with a latency of about 1 year in C57Bl/6J mice.5 In order to test whether Wt1 mutations can co-operate with PML-RARA in this model, we used CRISPR/Cas9 to generate indels in Wt1 exon 8 or, as a control, the Rosa26 locus. Since murine Wt1 is highly homologous to the human protein, these mutations should mimic those commonly found in APL patients. Despite efficient mutation generation in Ctsg-PML-RARA bone marrow cells (Online Supplementary Figure S2A), there was no survival difference between mice trans- planted with Ctsg-PML-RARA cells with Wt1 mutations versus Rosa26 mutations (Online Supplementary Figure S2C). We therefore evaluated APL tumors arising in these mice, and observed that tumors could arise either from wild-type or mutant Wt1/Rosa26 progenitors (Online Supplementary Figure S2B). Surprisingly, we did not detect Wt1 protein in these tumors by western blot- ting (data not shown). Similarly, in a panel of 16 previ- ously banked murine APL tumors from Ctsg-PML-RARA mice, RNA sequencing revealed virtually undetectable levels of Wt1 mRNA (Online Supplementary Figure S2D),
in contrast to the high expression seen in human APL samples. Together, these data suggest that important interspecies differences in Wt1 regulation and function are important for the lack of a phenotype in this mouse model.
We next evaluated WT1 expression in human CD34+ cells by transducing umbilical cord blood-derived CD34+ cells with retroviruses encoding PML-RARA, RUNX1-RUNX1T1, or MYC; RUNX1-RUNX1T1 and MYC have previously been shown to confer self-renewal and expansion of human HSPCs in vitro and in xenograft models, and therefore act as positive controls.6,7 Consistent with previously reported results, we found that human HSPC transduced with RUNX1-RUNX1T1 and MYC expand robustly over 2 weeks in culture, while HSPC transduced with PML-RARA expand more slowly (data not shown). In order to test whether WT1 expres- sion is affected by transduction with these retroviral constructs, GFP+ cells were sorted 7 days after transduc- tion, RNA was isolated, and WT1 expression was meas- ured by quantitative reverse transcription polymerase chain reaction (RT-PCR). Figure 1A shows upregulation of WT1 mRNA in sorted human HSPC transduced with PML-RARA, RUNX1-RUNX1T1, and MYC (6 to 18-fold increase, P<0.05 for PML-RARA and MYC). WT1 protein abundance also increased dramatically in the same cells during this timeframe (Figure 1B). In order to identify other genes dysregulated by PML-RARA transduction, we transduced both mouse and human HSPC with GFP- tagged retroviruses encoding PML-RARA or an empty vector, as has been previously reported (n=2 and n=3 separate experiments for human and mouse cells respec- tively).8,9 Seven days after transduction, GFP+ cells were sorted and bulk RNA sequencing was performed to iden- tify differentially expressed genes (DEG, 5,347 identified for mouse samples, and 1,885 identified for human sam- ples, Figure 1C). There was significant overlap between orthologous mouse and human DEG after transduction with PML-RARA (Figure 1D, P=9.6x10-118 based on the hypergeometric test); further, of 867 overlapping ortho- logues, 82% were coordinately regulated. However, while WT1 was ~13-fold upregulated in human cells transduced with PML-RARA, Wt1 expression was extremely low and did not increase in murine cells trans- duced with PML-RARA (Figure 1E), validating the inter- species difference in WT1/Wt1 regulation noted above.
GFP+ PML-RARA-expressing human cord blood cells expanded modestly during the first weeks of culture, but increased dramatically 3-4 weeks after transduction (Online Supplementary Figure S3A). After 6 weeks in cul- ture, they resembled primary APL cells morphologically and immunophenotypically (Online Supplementary Figure S3B and C). In addition, they were sensitive to treatment with all-trans retinoic acid (ATRA), a hallmark of APL cells (Online Supplementary Figure S3D). Transduced cells were not immortalized, as they stopped proliferating around 8-9 weeks after initiation, and they failed to engraft immunodeficient mice (data not shown). Given its reported role as a tumor suppressor in other cancer types, we hypothesized that this upregulation may reflect an attempt of WT1 to suppress the proliferative response induced by PML-RARA, similar to the increased TP53 activity observed in cells responding to genotoxic stressors.10 However, as noted above, it is also possible that high WT1 expression actively promotes the growth or survival of APL cells. In order to distinguish between these possibilities, we performed WT1 overexpression versus loss-of-function experiments in PML-RARA-trans- duced cord blood cells.
342
haematologica | 2022; 107(1)