Abstract
Mutations in the TP53 tumor suppressor gene are common in many cancer types, including the acute myeloid leukemia (AML) subtype known as complex karyotype AML (CK-AML). Here, we identify a gain-of-function (GOF) Trp53 mutation that accelerates CK-AML initiation beyond p53 loss and, surprisingly, is required for disease maintenance. The Trp53R172H mutation (TP53R175H in humans) exhibits a neomorphic function by promoting aberrant self-renewal in leukemic cells, a phenotype that is present in hematopoietic stem and progenitor cells (HSPC) even prior to their transformation. We identify FOXH1 as a critical mediator of mutant p53 function that binds to and regulates stem cell–associated genes and transcriptional programs. Our results identify a context where mutant p53 acts as a bona fide oncogene that contributes to the pathogenesis of CK-AML and suggests a common biological theme for TP53 GOF in cancer.
Our study demonstrates how a GOF p53 mutant can hijack an embryonic transcription factor to promote aberrant self-renewal. In this context, mutant Trp53 functions as an oncogene to both initiate and sustain myeloid leukemia and suggests a potential convergent activity of mutant Trp53 across cancer types.
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Introduction
TP53 mutations occur in the majority of human cancers and are often associated with poor outcomes (1, 2). TP53 encodes a sequence-specific transcription factor (3) that is normally maintained at low levels through strict post-translational control (4). In response to DNA damage, activated oncogenes, or other forms of cellular stress, p53 is stabilized and promotes cell-cycle arrest, apoptosis, senescence, or other antiproliferative programs depending on cellular context (5–8). Most TP53 mutations occur in the DNA binding domain and disrupt its transcriptional activity, thereby preventing these stress responses and enabling aberrant proliferation and survival of mutated cells (9).
Cancer-associated mutations typically inactivate p53 through a two-hit mechanism, whereby one allele acquires a missense mutation and the other undergoes “loss-of-heterozygosity” (LOH) via chromosomal deletion (8). Missense TP53 mutations encode proteins that have attenuated capacity to transactivate wild-type target genes, despite being frequently stabilized owing to reduced interaction with negative regulators (10). These mutant proteins can instill neomorphic gain-of-function (GOF) activities that contribute to cancer phenotypes beyond p53 loss (11). At the organismal level, mice harboring certain germline missense mutations in Trp53 develop an altered tumor spectrum compared with Trp53-null mice, including a larger fraction of epithelial cancers with increased metastatic potential (12, 13). At the cellular level, some GOF p53 mutants promote chemoresistance, invasiveness, and/or an epithelial-to-mesenchymal transition (EMT) through diverse mechanisms (12–14). Another neomorphic function of mutant p53 involves its ability to facilitate the formation of induced pluripotent stem cells (iPSC) more so than p53 loss (15, 16), although the extent to which this GOF activity is relevant to cancer is poorly understood.
In contrast to their high prevalence in most solid tumors, TP53 mutations occur in around 10% of blood cancers, although when they occur they are associated with poor prognosis (17, 18). In acute myeloid leukemia (AML), TP53 mutations are associated with a subtype known as complex karyotype AML (CK-AML), which is defined by the presence of three or more cytogenetic abnormalities and a dismal 5-year survival rate of less than 2% (17, 18). Functional studies in mice indicate that Trp53 inactivation in the hematopoietic compartment can produce chemoresistant malignancies with increased leukemia-initiating potential, mirroring key features linked to TP53 mutations in patients with AML (19–21). Still, whether and how TP53 missense mutations confer GOF activities to p53 in AML is not known.
In this study, we set out to test whether mutant p53 has GOF activity in AML and, if so, to determine the underlying mechanisms behind this effect. We chose to study Trp53R172H (R175H in humans), a mutant form of Trp53 that has been shown to confer GOF activity in solid tumors and is the most common allele in patients with AML (Dr. Elli Papaemmanuil, personal communication). Several complementary in vitro and in vivo systems were used to compare the biological features of wild-type, Trp53-null, or Trp53-mutant alleles, leading us to identify a neomorphic function of mutant p53 in hematopoietic stem and progenitor cells (HSPC) that exerts its effect by enhancing cellular self-renewal beyond that produced by p53 inactivation. We also identify a novel mediator of mutant p53 function, FOXH1, which contributes to the aberrant self-renewal phenotype. As such, suppression of either mutant p53 or FOXH1 ablates this stemness capacity by triggering differentiation. These observations illustrate how mutant p53 can acquire a pro-oncogenic activity that magnifies loss of its tumor-suppressive functions and creates a previously unappreciated molecular dependency in AML.
Results
p53R172H Accelerates the Onset of Hematologic Malignancies beyond Effects of p53 Deficiency
We first compared the ability of a mutant or null Trp53 allele to promote leukemogenesis in a well-defined genetic model. Because of its previously defined GOF activity in other settings, we used a conditional mutant Trp53 allele harboring an R172H mutation downstream of a “lox-stop-lox” cassette (LSL-Trp53R172H; ref. 12), which corresponds to TP53R175H, the most frequent missense mutation in clinical cases of AML (18). Mouse cohorts were produced to harbor one Trp53R172H allele and a Trp53 floxed allele (Trp53LSL-R172H/F), or were homozygous for the floxed Trp53 allele (Trp53F/F), such that all hematopoietic cells would become Trp53R172H/Δ and Trp53Δ/Δ, respectively, in the presence of the hematopoietic-specific Vav1-Cre transgene (22). Although both the Trp53R172H/Δ and Trp53Δ/Δ cohorts eventually succumbed to disease with full penetrance, mice harboring the Trp53R172H allele became moribund significantly faster (Fig. 1A). As commonly observed in Trp53−/− mice (23), most animals develop thymic lymphoma, although some had leukemias and, rarely, a mixed disease (leukemia and lymphoma; Fig. 1B).
Trp53R172H leads to accelerated onset of hematologic malignancies. A, Kaplan–Meier survival curves of native mice with Trp53Δ/Δ and Trp53R172H/Δ hematopoietic cells recombined using Vav1-Cre. B, Spectrum of hematologic malignancies arising in Trp53Δ/Δ (n = 15) and Trp53R172H/Δ (n = 11) native mice. C, Kaplan–Meier survival curves of thymectomized mice transplanted with Trp53f/f, Trp53f/f;Mx1-Cre and Trp53R172H/F;Mx1-Cre BM. D, Expression of CD11b and Gr1 in the peripheral blood of moribund mice from C. E, Representative hematoxylin and eosin image of mice with AML arising after transplant in thymectomized recipients. Black arrows show myeloid blasts.**, P < 0.005; ****, P < 0.0001, log-rank (Mantel–Cox) test.
Trp53R172H leads to accelerated onset of hematologic malignancies. A, Kaplan–Meier survival curves of native mice with Trp53Δ/Δ and Trp53R172H/Δ hematopoietic cells recombined using Vav1-Cre. B, Spectrum of hematologic malignancies arising in Trp53Δ/Δ (n = 15) and Trp53R172H/Δ (n = 11) native mice. C, Kaplan–Meier survival curves of thymectomized mice transplanted with Trp53f/f, Trp53f/f;Mx1-Cre and Trp53R172H/F;Mx1-Cre BM. D, Expression of CD11b and Gr1 in the peripheral blood of moribund mice from C. E, Representative hematoxylin and eosin image of mice with AML arising after transplant in thymectomized recipients. Black arrows show myeloid blasts.**, P < 0.005; ****, P < 0.0001, log-rank (Mantel–Cox) test.
The above observations are consistent with those previously obtained using mice harboring a humanized TP53R248Q allele expressed in the whole body of mice, which also develop T-cell lymphoma faster than Trp53-null mice (24). However, neither model addresses the biology of mutant p53 action in the clinically relevant context of AML, likely due to the high penetrance of lymphomas that arise in the mouse. Therefore, to bias the model against T-cell lymphoma development, we transplanted bone marrow (BM) cells from Mx1-Cre;Trp53R172H/F and Mx1-Cre;Trp53F/F mice into thymectomized recipients. Three weeks post-transplantation, intraperitoneal injection of PolyI:C was used to activate the IFN-inducible Mx1-Cre, triggering the recombination of floxed alleles in transplanted cells (25). Of note, the use of the inducible Mx1-Cre recombinase enabled transplantation of functionally wild-type whole bone marrow (WBM) cells, avoiding biases in engraftment efficiency that might occur upon p53 alteration. As in the autochthonous model, Trp53R172H/Δ mice succumbed to disease faster than their Trp53-null counterparts (Fig. 1C), although, in this instance, the disease was immunophenotypically and histologically characterized as AML based on the expression of CD11b and Gr1 on the surface of infiltrating leukemic cells (Fig. 1D and E). Thus, p53R172H gives rise to an aggressive leukemia that significantly shortens overall survival as compared with the disease arising from p53-deficient BM cells, providing functional evidence that a Trp53-mutant allele can contribute to AML beyond the effects of p53 loss.
Sustained Expression of Mutant p53 Is Required for the Maintenance of p53R172H Leukemic Cells
We next examined whether the p53R172H-mutant protein was required for survival of p53-mutant leukemic cells. We utilized previously characterized Trp53Δ/Δ or Trp53R172H/Δ and Trp53R172H/11B3 CK-AML murine cell lines that were created by cotransduction of short hairpin RNAs (shRNA) targeting Nf1 (GFP-labeled) and Mll3 (mCherry-labeled), hereafter referred to as PNM (20, 26). These cell lines were generated to reflect the most common genetic configurations found in patients, including Trp53 loss alone (Trp53Δ/Δ), Trp53R172H over a focal Trp53 deletion (Trp53R172H/Δ), or Trp53R172H over a larger genomic deletion that encompasses Trp53 (Trp53R172H/11B3; ref. 25). Control shRNAs against Renilla luciferase (sh.Ren) or Trp53 (sh.p53) were coexpressed with a blue fluorescent protein (BFP) reporter (Supplementary Fig. S1A; ref. 27). The percentage of BFP+ cells was monitored over time as a measurement of fitness under the two shRNA-containing conditions.
We observed a significant reduction in the relative number of sh.p53 BFP+ cells compared with sh.Ren BFP+ cells over time (Fig. 2A, light blue and dark blue lines), indicating negative selection against cells in which the mutant protein is depleted. In contrast, sh.p53 had no effect on Trp53Δ/Δ AML cells, ruling out potential off-target effects of the p53 shRNA (Fig. 2A, black line). p53 depletion in cells harboring Trp53R172H promoted differentiation and triggered apoptosis as assessed by flow cytometry for CD11b and Annexin V, respectively (Fig. 2B and C). Thus, AML cells expressing p53R172H acquire a molecular dependency on mutant p53 that contributes to a differentiation block that sustains leukemogenesis.
Depletion of mutant p53 impairs the growth of p53-mutant leukemias. A, The ratio of sh.p53 BFP+ cells compared with sh.Ren BFP+ cells over time in leukemic lines. CD11b expression in the shRNA BFP+ containing cells (n = 3; B) and Annexin V (C) shown by FACS 7 days after infection. Data presented as mean ± SEM. D, Relative expression of TP53 7 days postinfection with pRS sh.TP53 in human KY821 cells. E, Colony-forming units (CFU) in human AML cells with sh.Ren or sh.TP53. Data presented as mean ± SEM (n = 3). F, hCD16 expression by FACS in pRS Ctrl or pRS sh.TP53 cells from growing in CFU assay. G, Percentage of hCD45 in the peripheral blood of mice 5 weeks post-transplant in shCtrl and shCtrl moribund mice (black),sh.TP53 (blue), and sh.TP53 moribund (orange) mice. Data are represented as mean ± SEM (n = 5 and n = 5). H, Kaplan–Meier curve from NSG micetransplanted with equal numbers of KY821 cells transduced with either shCtrl or sh.TP53 (n = 5 and n = 5). I, Western blot analysis for human p53 in cells before injection and in sorted hCD45 cells from the peripheral blood of three independent moribund sh.TP53 mice. B, Kruskal–Wallis one-way ANOVA; C, D, E, H, unpaired t test. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.
Depletion of mutant p53 impairs the growth of p53-mutant leukemias. A, The ratio of sh.p53 BFP+ cells compared with sh.Ren BFP+ cells over time in leukemic lines. CD11b expression in the shRNA BFP+ containing cells (n = 3; B) and Annexin V (C) shown by FACS 7 days after infection. Data presented as mean ± SEM. D, Relative expression of TP53 7 days postinfection with pRS sh.TP53 in human KY821 cells. E, Colony-forming units (CFU) in human AML cells with sh.Ren or sh.TP53. Data presented as mean ± SEM (n = 3). F, hCD16 expression by FACS in pRS Ctrl or pRS sh.TP53 cells from growing in CFU assay. G, Percentage of hCD45 in the peripheral blood of mice 5 weeks post-transplant in shCtrl and shCtrl moribund mice (black),sh.TP53 (blue), and sh.TP53 moribund (orange) mice. Data are represented as mean ± SEM (n = 5 and n = 5). H, Kaplan–Meier curve from NSG micetransplanted with equal numbers of KY821 cells transduced with either shCtrl or sh.TP53 (n = 5 and n = 5). I, Western blot analysis for human p53 in cells before injection and in sorted hCD45 cells from the peripheral blood of three independent moribund sh.TP53 mice. B, Kruskal–Wallis one-way ANOVA; C, D, E, H, unpaired t test. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.
To determine whether human AML can also depend on sustained expression of mutant p53, we first examined publicly available CRISPR/Cas9 genome-wide screening data (28). In these studies, single guide RNAs (sgRNA) were introduced into human AML lines expressing Cas9 followed by assessment of their enrichment or depletion over time using next-generation sequencing. Interestingly, sgRNAs targeting p53 were invariably enriched in AML lines harboring wild-type TP53 but depleted to varying degrees in those expressing mutant TP53 genes (e.g., R248Q, another hotspot TP53 mutation), suggesting that a subset of the latter group might depend on sustained expression of the mutant protein for their proliferation and/or survival (Supplementary Fig. S1B).
None of the AML cell lines used in the aforementioned CRISPR/Cas9 screen harbored an R175H mutation (corresponding to the mutation we studied in mice). However, we identified a human AML cell line (KY821) that harbored this mutation in the absence of a wild-type (WT) TP53 allele (Supplementary Fig. S1C) and used it to functionally test the requirement for this mutant form of TP53 in vitro and in vivo. Cells were transduced with a validated shRNA targeting human p53 (sh.TP53; ref. 29) or empty-vector control (Ctrl) and examined for their ability to form colonies in methylcellulose culture in vitro or give rise to leukemia upon transplantation into sublethally irradiated NOD-SCID IL2R−/− (NSG) immunodeficient mice. Similar to observations from murine AML, suppression of mutant TP53 in KY821 cells significantly reduced their colony-forming ability (Fig. 2D and E) and led to the upregulation of the myeloid differentiation marker CD16 (Fig. 2F), again suggesting that this p53 mutant contributes to a differentiation block.
To test whether TP53R175H-mutant cells transduced with sh.TP53 exhibited reduced fitness in vivo, we analyzed peripheral blood for the presence of human CD45-positive leukocytes in recipient mice 5 weeks post-transplantation. Remarkably, this analysis revealed a significant reduction of cells containing sh.TP53 versus control cells (Fig. 2G), confirming the suppressive effect of mutant p53 knock-down in p53 mutant–expressing human leukemias. In addition, knockdown of mutant p53 significantly increased the survival of transplanted mice compared with controls (Fig. 2H). At the time of death, the resulting leukemias invariably selected against expression of the p53-targeting shRNA and restored the expression of mutant p53, consistent with our observations from murine models (Fig. 2I; Supplementary Fig. S1D–S1F). Therefore, KY821 cells require mutant p53 protein to sustain leukemia. Collectively, these results reveal an oncogenic function for mutant p53 in murine and human AML.
p53R172H-Derived Leukemia Is Associated with Stem Cell Transcriptional Signatures
Most studies suggest that the GOF activities of mutant p53 proteins arise from their ability to alter transcription in a manner that is distinct from effects due to p53 loss (30). Therefore, to examine how mutant p53 altered the transcriptome of CK-AML cells, we performed gene expression profiling of PNM Trp53-null and Trp53-mutant leukemias via RNA sequencing (RNA-seq) and identified 252 significantly differentially expressed genes (Fig. 3A; Supplementary Table S1). A systematic and unbiased analysis of the data using gene ontology (GO) pathway analysis revealed TGFβ and stem cell pathways to be positively correlated with the top upregulated genes in our Trp53R172H murine leukemias (Fig. 3B). Notably, these same signatures were enriched in the transcriptional profiles of leukemias obtained from patients with CK-AML, the AML subtype in which missense TP53 mutations most commonly occur, compared with other leukemia subtypes or normal hematopoietic cells (www.bloodspot.eu; ref. 32; Fig. 3C). Beyond general stem cell signatures, gene set enrichment analysis (GSEA) indicated that p53-mutant AML was enriched for genes linked to hematopoietic stem cells (HSC) and long term-HSC (LT-HSC; ref. 31; Fig. 3D). Therefore, both murine and human mutant p53-expressing (but not p53-deficient) leukemias converge on similar transcriptional pathways, some of which are linked to stemness and inhibition of myeloid differentiation.
Gene expression profile of Trp53R172H/Δ leukemias. A, Volcano plot from RNA-seq comparing Trp53R172H/Δ leukemias to Trp53Δ/Δ showing Trp53 as the most highly expressed gene between the two groups. B, Gene ontology (GO) analysis of pathways associated with genes upregulated in Trp53R172H/Δ leukemias. C, Expression of GO-associated pathways in human AML samples and normal human hematopoietic cells (www.bloodspot.eu). D, Gene set enrichment analysis comparing gene expression between Trp53R172H/Δ and Trp53Δ/Δ leukemias to other known signatures related to stem cells and hematopoietic stem cells (HSC). Two-tailed t test, *, P < 0.05; **, P < 0.005; ***, P < 0.0005. MPP, multipotential progenitors; early_PM, early promyelocyte; late_PM, late promyelocyte; MY, myelocyte; MM, metamyelocytes; BC, band cell; PMN, polymorphonuclear cells; mono, monocytes; LT-HSC, long-term HSC.
Gene expression profile of Trp53R172H/Δ leukemias. A, Volcano plot from RNA-seq comparing Trp53R172H/Δ leukemias to Trp53Δ/Δ showing Trp53 as the most highly expressed gene between the two groups. B, Gene ontology (GO) analysis of pathways associated with genes upregulated in Trp53R172H/Δ leukemias. C, Expression of GO-associated pathways in human AML samples and normal human hematopoietic cells (www.bloodspot.eu). D, Gene set enrichment analysis comparing gene expression between Trp53R172H/Δ and Trp53Δ/Δ leukemias to other known signatures related to stem cells and hematopoietic stem cells (HSC). Two-tailed t test, *, P < 0.05; **, P < 0.005; ***, P < 0.0005. MPP, multipotential progenitors; early_PM, early promyelocyte; late_PM, late promyelocyte; MY, myelocyte; MM, metamyelocytes; BC, band cell; PMN, polymorphonuclear cells; mono, monocytes; LT-HSC, long-term HSC.
p53R172H Enhances Self-Renewal in Hematopoietic Cells In Vitro and In Vivo
AML arises from alterations (e.g., mutations) that promote proliferation or enhance the self-renewal of myeloid progenitor cells (33, 34). To directly examine the functional consequences of mutant p53 expression in the normal hematopoietic compartment, we isolated adult WBM cells from Trp53WT/WT, Trp53Δ/Δ, and Trp53R172H/Δ mice and performed limiting dilution cultures in methylcellulose medium followed by serial replating. Under these cytokine-rich culture conditions, wild-type cells lose their replating capacity after three sequential passages (35). As described previously (36), loss of p53 modestly increased the replating capacity of hematopoietic cells as compared with those expressing wild-type p53 (Fig. 4A). In stark contrast, hematopoietic cells expressing Trp53R172H/Δ not only replated more frequently than Trp53Δ/Δ and WT controls, but also could replate indefinitely (Fig. 4A).
p53R172H induces aberrant self-renewal in vitro and in vivo. A, Total number of colony-forming units (CFU) generated by Trp53WT (black), Trp53Δ/Δ (red) and Trp53R172H/Δ (blue) cells. Data are representative of four independent experiments. Error bars correspond to mean ± SEM (n = 3). B, Total number of CFUs generated by Trp53R172H/Δ c-KIT+ cells containing sh.Ren or sh.p53. Data are representative of three independent experiments. Error bars correspond to mean ± SEM (n = 3). C, Western blotting for p53 and β-Actin at passage 4 (P4). D, Schematic representation of the competitive transplantation protocol. E, Percentage of CD45.2 cells during the secondary competitive transplant in the peripheral blood (PB) of mice monitored monthly by bleeding (n = 5 per group). F, Percentage of CD45.2 cells in the PB four months after transplant (n = 4–7). G, Percentage of CD45.2 cells in the BM 4 months after transplant (n = 4–7). H, Percentage of megakaryocyte erythrocyte progenitors (MEP), granulocyte monocyte progenitors (GMP; I) and common myeloid progenitors (CMP; J) in the BM of mice four months after transplant (n = 4–7). K, Schematic representation of the transplant layout. LSKs were sorted from Trp53R172H/Δ mice and were infected with an sh.Ren or sh.p53 conjugated to BFP fluorescence. Two days after infection equal numbers of cells were transplanted in lethally irradiated CD45.1 mice. L, BFP fluorescence over time as assessed by monthly bleeds in recipient mice (n = 5). M, Percentage of BFP+ cells in the BM of mice with sh.Ren or sh.p53 4 months after transplant (n = 3–5). N, Percentage of BFP+ LSKs in the BM of mice with sh.Ren or sh.p53 4 months after transplant (n = 3–5). B, C, and D, two-tailed t test, *, P < 0.05; **, P < 0.005; ***, P < 0.0005. Error bars correspond to mean ± SEM.
p53R172H induces aberrant self-renewal in vitro and in vivo. A, Total number of colony-forming units (CFU) generated by Trp53WT (black), Trp53Δ/Δ (red) and Trp53R172H/Δ (blue) cells. Data are representative of four independent experiments. Error bars correspond to mean ± SEM (n = 3). B, Total number of CFUs generated by Trp53R172H/Δ c-KIT+ cells containing sh.Ren or sh.p53. Data are representative of three independent experiments. Error bars correspond to mean ± SEM (n = 3). C, Western blotting for p53 and β-Actin at passage 4 (P4). D, Schematic representation of the competitive transplantation protocol. E, Percentage of CD45.2 cells during the secondary competitive transplant in the peripheral blood (PB) of mice monitored monthly by bleeding (n = 5 per group). F, Percentage of CD45.2 cells in the PB four months after transplant (n = 4–7). G, Percentage of CD45.2 cells in the BM 4 months after transplant (n = 4–7). H, Percentage of megakaryocyte erythrocyte progenitors (MEP), granulocyte monocyte progenitors (GMP; I) and common myeloid progenitors (CMP; J) in the BM of mice four months after transplant (n = 4–7). K, Schematic representation of the transplant layout. LSKs were sorted from Trp53R172H/Δ mice and were infected with an sh.Ren or sh.p53 conjugated to BFP fluorescence. Two days after infection equal numbers of cells were transplanted in lethally irradiated CD45.1 mice. L, BFP fluorescence over time as assessed by monthly bleeds in recipient mice (n = 5). M, Percentage of BFP+ cells in the BM of mice with sh.Ren or sh.p53 4 months after transplant (n = 3–5). N, Percentage of BFP+ LSKs in the BM of mice with sh.Ren or sh.p53 4 months after transplant (n = 3–5). B, C, and D, two-tailed t test, *, P < 0.05; **, P < 0.005; ***, P < 0.0005. Error bars correspond to mean ± SEM.
This enhanced capability could not be explained by differences in the preexisting frequency of stem and progenitor cells in the BM, because immunophenotyping of 7-week-old mice (the time of BM collection) did not show an increase in any type of stem or progenitor cell (Supplementary Fig. S2A–S2K). This increase in self-renewal was observed in Trp53R172H/Δ but not Trp53R172H/WT cells, suggesting that loss of the residual WT allele is a prerequisite for this GOF effect (Supplementary Fig. S3A). Furthermore, Trp53R172H/Δ cells exhibited higher nuclear-to-cytoplasmic ratio when compared with Trp53Δ/Δ and WT cells at the same passage (P3; Supplementary Fig. S3B) and expressed the highest levels of CD34 (Supplementary Fig. S3C), an important marker of long-term repopulation of HSCs (37). The replating phenotype produced by mutant Trp53 was similar to that produced by disruption of Tet2, a well-characterized leukemia tumor suppressor gene whose loss promotes enhanced self-renewal (Supplementary Fig. S3C; refs. 38, 39). Nevertheless, the Trp53R172H/Δ cells were different than Tet2Δ/Δ cells at the immunophenotypic and morphologic levels (Supplementary Fig. S3C and S3D), suggesting subtle differences between how these alterations act.
To further explore the relationship between mutant p53 and self-renewal potential, we isolated c-KIT+ BM cells from the Trp53R172H/Δ mice and transduced them with viral vectors coexpressing an shRNA-targeting mutant p53 (sh.p53) or a Renilla control (sh.Ren). These vectors encoded puromycin resistance, allowing us to select and maintain pure shRNA-expressing cells in culture. This isogenic comparison validated our previous observation: p53 knockdown in Trp53-mutant cells halted replating capacity after the fourth passage (comparable with Trp53Δ/Δ WBM), whereas sh.Ren cells (which retain expression of mutant p53) were able to replate indefinitely (Fig. 4B and C). We confirmed that the normalization of self-renewal potential by sh.p53 was not due to an off-target effect of RNAi, as the same shRNA had no effect on the fitness of Trp53Δ/Δ hematopoietic cells (Supplementary Fig. S2L).
Consistent with these findings, competitive transplantation studies using Trp53WT/WT, Trp53Δ/Δ, and Trp53R172H/Δ BM (Fig. 4D) demonstrated that Trp53R172H/Δ cells outcompeted wild-type cells to a greater extent than Trp53Δ/Δ cells (Supplementary Fig. S3E–S3G), a phenotype that was further exacerbated in secondary transplants (Fig. 4E). Analysis of peripheral blood (Fig. 4F) and BM (Fig. 4G) demonstrated that Trp53R172H/Δ cells were significantly enriched with respect to Trp53WT cells. Furthermore, within the BM of Trp53R172H/Δ mice, HSCs were expanded in relative and absolute number compared with Trp53Δ/Δ mice (Supplementary Fig. S3H–S3K). All downstream progenitors, including megakaryocyte/erythroid progenitors (MEP), granulocyte/macrophage progenitors (GMP) and common myeloid progenitors (CMP), were significantly increased in Trp53R172H/Δ mice (Fig. 4H–J).
For further validation, we examined the consequences of inhibiting mutant p53 in normal mouse HSCs. For these experiments, we used the BM of Trp53R172H/Δ mice to isolate lineage-negative (Lin−)/Sca1+/c-KIT+ cells (LSK), a cell population capable of maintaining long-term hematopoiesis in mice (40). To suppress p53, these cells were infected with retroviruses encoding an shRNA (targeting mutant p53 or Renilla control) linked to BFP (Fig. 4K). Equal numbers of either sh.Ren or sh.p53 cells were mixed with non–shRNA-expressing p53R172H BFP− cells and transplanted into CD45.1 lethally irradiated mice in an in vivo competition format. The fraction of BFP+ cells in the peripheral blood was monitored for 4 months. Trp53R172H/Δ BM cells transduced with the p53 shRNA were progressively depleted from peripheral blood, becoming nearly undetectable by the end of the experiment (Fig. 4L).
Analysis of BM at the final time point revealed that fewer cells expressing sh.p53 remained as compared with those expressing control sh.Ren (Fig. 4M). Moreover, the proportion of LSKs within the sh.p53 population was lower than in the control sh.Ren population (Fig. 4N). Altogether, these observations establish a role for p53R172H in promoting aberrant self-renewal in adult hematopoietic cells in a manner that renders these cells dependent on its action for their maintenance in a premalignant setting. Hence, phenotypes linked to p53-mutant GOF can emerge in the hematopoietic system prior to neoplastic transformation.
FOXH1 Is Required for Mutant p53–Induced Self-Renewal in Both Normal and Leukemic Cells
To explore the mechanism by which mutant p53 enhances cellular self-renewal, we surveyed the genes that were expressed at higher levels in Trp53R172H/Δ leukemias compared with Trp53Δ/Δ leukemias. One of the most significantly upregulated genes in our analysis was Foxh1 (Supplementary Table S1), which encodes a key transcription factor that is known to mediate Nodal/TGFβ signaling during embryogenesis, but is not expressed at appreciable levels in the adult hematopoietic compartment or any other mouse tissue (Supplementary Fig. S4A–S4C). Like mutant p53 (41), FOXH1 has been linked to EMT (42, 43) and can facilitate the reprogramming of fibroblasts into iPSCs (44). Furthermore, many of the top upregulated genes in Trp53R172H/Δ leukemias were known FOXH1 targets (ref. 45; Fig. 5A). Taken together, these observations led us to hypothesize that FOXH1 might be a key mediator of the p53R172H GOF activity in leukemia.
Identification of FOXH1 as a p53-mutant mediator. A, GSEA comparing the expression of genes associated with genomic regions occupied by FOXH1 when compared with the genes differentially expressed in Trp53R172H and Trp53Δ/Δ leukemias. B, Microarray data showing the expression of FOXH1 across normal human hematopoietic cells and different types of leukemias. C, Overall survival in patients with AML who have FOXH1 mRNA upregulation or genetic amplification compared with patients without (cBioPortal –TCGA). D, Western blotting for FOXH1 in HSPCs from Trp53R172H and Trp53Δ/Δ cells at Passage 4. E, Western blotting for p53 and FOXH1 in Trp53R172H HSPCs transduced with sh.Ren or sh.p53. F, Western blotting for TP53 and FOXH1 in two independent Trp53R172H murine leukemic cells transduced with sh.Ren or sh.p53. G, Western blotting for p53 and FOXH1 in Trp53R175H human leukemic cells transduced with sh.Ren or sh.TP53. *, P < 0.05; ***, P < 0.0005. MPP, multipotential progenitors; CMP, common myeloid progenitor cell; GMP, granulocyte monocyte progenitors; MEP, megakaryocyte-erythroid progenitor cell; early_PM, early promyelocyte; late_PM, late promyelocyte; MY, myelocyte; MM, metamyelocytes; BC, band cell; PMN, polymorphonuclear cells; mono, monocytes.
Identification of FOXH1 as a p53-mutant mediator. A, GSEA comparing the expression of genes associated with genomic regions occupied by FOXH1 when compared with the genes differentially expressed in Trp53R172H and Trp53Δ/Δ leukemias. B, Microarray data showing the expression of FOXH1 across normal human hematopoietic cells and different types of leukemias. C, Overall survival in patients with AML who have FOXH1 mRNA upregulation or genetic amplification compared with patients without (cBioPortal –TCGA). D, Western blotting for FOXH1 in HSPCs from Trp53R172H and Trp53Δ/Δ cells at Passage 4. E, Western blotting for p53 and FOXH1 in Trp53R172H HSPCs transduced with sh.Ren or sh.p53. F, Western blotting for TP53 and FOXH1 in two independent Trp53R172H murine leukemic cells transduced with sh.Ren or sh.p53. G, Western blotting for p53 and FOXH1 in Trp53R175H human leukemic cells transduced with sh.Ren or sh.TP53. *, P < 0.05; ***, P < 0.0005. MPP, multipotential progenitors; CMP, common myeloid progenitor cell; GMP, granulocyte monocyte progenitors; MEP, megakaryocyte-erythroid progenitor cell; early_PM, early promyelocyte; late_PM, late promyelocyte; MY, myelocyte; MM, metamyelocytes; BC, band cell; PMN, polymorphonuclear cells; mono, monocytes.
In agreement, analysis of publicly available expression data from normal human hematopoietic cells and different leukemia subtypes revealed that FOXH1 expression was significantly higher in all AMLs (particularly in patients with CK-AML, the majority of whom harbor TP53 mutations) compared with any type of normal cell (Fig. 5B; Supplementary Fig. S5A and S5B). In addition, like mutant TP53, FOXH1 upregulation correlated with poor survival in patients with AML (refs. 46, 47; Fig. 5C). Consistent with the RNA-seq results, FOXH1 expression was also higher in Trp53R172H/Δ HSPCs compared with Trp53Δ/Δ cells growing in colonies (Fig. 5D), and knockdown of mutant p53 reduced FOXH1 levels in murine HSPCs (Fig. 5E) as well as murine and human leukemic cells (Fig. 5F and G, respectively). Thus, mutant p53 expression led to higher levels of FOXH1 in both normal and leukemic cells from mouse and human leukemias.
FOXH1 Is a Critical Effector of p53R172H via Its Ability to Bind and Regulate Stem Cell Genes
Because FOXH1 is a transcription factor and its role in cells of hematopoietic origin is not known, we performed chromatin immunoprecipitation (ChIP) with an anti-FOXH1 antibody followed by high-throughput sequencing (ChIP-seq) to identify the genome-wide FOXH1 binding sites in Trp53-mutant murine leukemic cells (Fig. 6A). Sites bound by FOXH1 correlated with the presence of canonical FOXH1 binding motifs (P < 1e-1915) as well as with other TGFβ mediators (refs. 48, 49; Fig. 6B). GO pathway analysis indicated that genes associated with FOXH1 peaks are involved in BMP and TGFβ signaling pathways, as well as myeloid leukemia (Fig. 6C).
FOXH1 directly binds genes involved in leukemia and stem cell properties. A, Pie charts depicting the genome-wide distribution of FOXH1 binding in murine leukemic cells. TSS, transcription start site. B, Motif enrichment analysis for the FOXH1-bound genomic regions in leukemic cells. C, GO analysis of genes bound by FOXH1 in murine leukemic cells. D, Venn diagram depicting the overlap between the genes downregulated upon p53 KD (blue circle), FOXH1 KD (green circle), and FOXH1 ChIP (orange circle). E, GSEA from transcriptional profile of cells upon FOXH1 KD. F, Gene tracks for FOXH1 ChIP-seq at the Runx2 and Mef2c loci together with RNA-seq tracks from either sh.Ren (red), sh.FOXH1 (green), or sh.p53 (blue). Mean log2 expression of transcriptional signature composed of significantly upregulated genes in Trp53R172H/Δ versus Trp53Δ/Δ murine leukemias across AML and normal cells (G), as well as same comparison for genes downregulated following FOXH1 KD (H). Student t test between CK-AML and other AML types, *, P < 0.05; ****, P < 0.00005.
FOXH1 directly binds genes involved in leukemia and stem cell properties. A, Pie charts depicting the genome-wide distribution of FOXH1 binding in murine leukemic cells. TSS, transcription start site. B, Motif enrichment analysis for the FOXH1-bound genomic regions in leukemic cells. C, GO analysis of genes bound by FOXH1 in murine leukemic cells. D, Venn diagram depicting the overlap between the genes downregulated upon p53 KD (blue circle), FOXH1 KD (green circle), and FOXH1 ChIP (orange circle). E, GSEA from transcriptional profile of cells upon FOXH1 KD. F, Gene tracks for FOXH1 ChIP-seq at the Runx2 and Mef2c loci together with RNA-seq tracks from either sh.Ren (red), sh.FOXH1 (green), or sh.p53 (blue). Mean log2 expression of transcriptional signature composed of significantly upregulated genes in Trp53R172H/Δ versus Trp53Δ/Δ murine leukemias across AML and normal cells (G), as well as same comparison for genes downregulated following FOXH1 KD (H). Student t test between CK-AML and other AML types, *, P < 0.05; ****, P < 0.00005.
To investigate how many of the genes bound by FOXH1 are dynamically regulated, we suppressed FOXH1 and compared the downregulated genes (green circle) to those physically bound by FOXH1 (orange circle; Fig. 6D). Consistent with a molecular relationship between mutant p53 and FOXH1 expression, there was a significant overlap between the genes that were decreased upon knockdown of mutant p53 (blue circle) or FOXH1 (green circle), with more than 50% of genes showing evidence of coregulation (Fisher exact test P < 2.2e-16). GSEA confirmed that genes that were downregulated upon FOXH1 knockdown significantly overlapped with those downregulated upon mutant p53 knockdown (NES = −14.5, FDR = 0; Fig. 6E). In line with these results, many of the transcriptional signatures related to HSCs and leukemia (previously shown to be upregulated in Trp53R172H/Δ cells) were conversely enriched upon suppression of either FOXH1 (Fig. 6E) or p53 (Supplementary Fig. S6A). As examples, two genes involved in leukemia and hematopoietic cell differentiation, Runx2 (50) and Mef2c (51), are both bound by FOXH1 and their expression is reduced by either FOXH1 or p53 knockdown (Fig. 6F). Hence, our data show that mutant p53 induces FOXH1, which can directly bind to and control genes linked to the differentiation status and self-renewal properties of AML.
To confirm that these FOXH1-dependent transcripts were also observed in human AML, we derived a transcriptional signature composed of differentially upregulated genes in Trp53R172H/Δ versus Trp53Δ/Δ murine leukemias and compared it with expression data from different karyotypes of human AML as well as normal hematopoietic cells (32). We observed a significant upregulation of the Trp53R172H/Δ murine leukemia–derived transcriptional signature in patients with CK-AML (dark red) compared with all other leukemia types (light red) or normal hematopoietic cells (black; Fig. 6G), suggesting our model transcriptionally resembles CK-AML harboring TP53 mutations. A similar increase in FOXH1-dependent transcripts (sh.FOXH1 vs. sh.Ren) was also observed in patients with CK-AML (Fig. 6H). These results reveal a FOXH1 signature in human AML harboring TP53 mutations, further supporting the existence of a functional mutant p53–FOXH1 axis in this disease.
FOXH1 Is Necessary and Sufficient for the Enhanced Self-Renewal Phenotype Produced by Mutant p53
To further explore the links between mutant p53, FOXH1, and aberrant self-renewal, we knocked down mutant p53 or FOXH1 in Trp53R172H/Δ HSPCs and performed colony-forming unit (CFU) and immunophenotyping assays. Knockdown of FOXH1 ablated the replating capacity of these cells (Fig. 7A), phenocopying, in large part, the effects observed upon mutant p53 knockdown. Furthermore, similar to mutant p53 ablation, FOXH1 suppression leads to the downregulation of the stem cell factor receptor c-KIT and the stem cell antigen 1 (Sca-1) in Trp53R172H/Δ HSPCs (but not in p53-deficient cells), which was functionally accompanied by coarsening of chromatin and nuclear segmentation, morphologic changes indicative of differentiation (Fig. 7B; Supplementary Fig. S7A and S7B). Conversely, overexpression of FOXH1 in both p53WT/WT and p53Δ/Δ HSPCs endowed these cells with increased colony-forming ability (Fig. 7C) and also led to the upregulation of c-KIT and Sca-1 along with concomitant downregulation of Gr1/CD11b (Fig. 7D; Supplementary Fig. S7C). Analogous results were also observed in leukemia cells, where suppression of either mutant p53 or FOXH1 in two Trp53-mutant AML lines reduced c-KIT expression (Supplementary Fig. S8A–S8D) and led to cellular differentiation determined by morphologic criteria (Supplementary Fig. S8E and S8F) and transcriptional profiling (Supplementary Fig. S8G). Finally, to determine whether FOXH1 could phenocopy mutant p53 effects, we performed an epistasis experiment using Trp53R172H/Δ HSPCs where mutant p53 was suppressed and cells were cotransduced to overexpress either a Foxh1 cDNA or control vector and examined for self-renewal capacity in serial replating assays. Enforced FOXH1 expression partially reversed the loss of replating potential produced by suppressing mutant p53 (Fig. 7E), suggesting that FOXH1 functions downstream of mutant p53 to enforce aberrant self-renewal. Collectively, these data demonstrate that FOXH1 is necessary and sufficient to support an aberrant self-renewal phenotype in both premalignant and malignant p53-mutant cells.
FOXH1 is necessary and sufficient for the enhanced self-renewal phenotype produced by mutant p53. A, Total number of CFUs generated by Trp53R172H/Δ HSPCs containing sh.p53 (blue), sh.Ren (black), shFOXH1.1072 (1; plum), shFOXH1.1116 (2; maroon). Data are representative of three experiments. Error bars correspond to mean ± SEM (n = 3). Bottom, Western blotting intensities for FOXH1 for the different conditions. B, c-KIT+ and Sca-1+ expression analyzed by FACS in sh.Ren, sh.p53 and sh.FOXH1 containing HSPCs from Trp53WT, Trp53Δ/Δ, and Trp53R172H/Δ mice. Data are representative of three experiments. C, Total number of CFUs generated by Trp53Δ/Δ and Trp53WT HSPCs containing pLVX Control (black) or pLVX Foxh1 cDNA (gray). Data are representative of three experiments. Error bars correspond to mean ± SEM (n = 3). Bottom, Western blotting for FOXH1 for the different conditions. D, Percentage of c-KIT+Sca-1+ HSPCs analyzed by FACS in Trp53Δ/Δ and Trp53WT cells containing pLVX Control or pLVX Foxh1 cDNA. Data are representative of three experiments. Error bars correspond to mean ± SEM (n = 3). E, Total number of CFUs generated by Trp53R172H/Δ HSPCs containing sh.Ren + pLVX control (black), sh.Ren + pLVX Foxh1 cDNA (gray), sh.p53 + pLVX control (light blue), sh.p53 + pLVX Foxh1 cDNA (dark blue). Data are representative of three experiments. Error bars correspond to mean ± SEM (n = 3; *, P < 0.05; **, P < 0.005; ***, P < 0.0005).
FOXH1 is necessary and sufficient for the enhanced self-renewal phenotype produced by mutant p53. A, Total number of CFUs generated by Trp53R172H/Δ HSPCs containing sh.p53 (blue), sh.Ren (black), shFOXH1.1072 (1; plum), shFOXH1.1116 (2; maroon). Data are representative of three experiments. Error bars correspond to mean ± SEM (n = 3). Bottom, Western blotting intensities for FOXH1 for the different conditions. B, c-KIT+ and Sca-1+ expression analyzed by FACS in sh.Ren, sh.p53 and sh.FOXH1 containing HSPCs from Trp53WT, Trp53Δ/Δ, and Trp53R172H/Δ mice. Data are representative of three experiments. C, Total number of CFUs generated by Trp53Δ/Δ and Trp53WT HSPCs containing pLVX Control (black) or pLVX Foxh1 cDNA (gray). Data are representative of three experiments. Error bars correspond to mean ± SEM (n = 3). Bottom, Western blotting for FOXH1 for the different conditions. D, Percentage of c-KIT+Sca-1+ HSPCs analyzed by FACS in Trp53Δ/Δ and Trp53WT cells containing pLVX Control or pLVX Foxh1 cDNA. Data are representative of three experiments. Error bars correspond to mean ± SEM (n = 3). E, Total number of CFUs generated by Trp53R172H/Δ HSPCs containing sh.Ren + pLVX control (black), sh.Ren + pLVX Foxh1 cDNA (gray), sh.p53 + pLVX control (light blue), sh.p53 + pLVX Foxh1 cDNA (dark blue). Data are representative of three experiments. Error bars correspond to mean ± SEM (n = 3; *, P < 0.05; **, P < 0.005; ***, P < 0.0005).
Discussion
Although mutations in the TP53 tumor suppressor gene ablate its antiproliferative functions, some mutant TP53 alleles also appear to produce “GOF” phenotypes that display more profound phenotypes than those produced by complete p53 loss. Nonetheless, studies investigating the basis for these effects have failed to coalesce around a common biological or molecular mechanism and, indeed, a recent comprehensive analysis of DNA sequence and functional genomics data suggested that the primary selective pressure for the appearance of p53 mutation in cancer arises from their purely inactivating and/or dominant-negative effects (52). Here we studied the action of the most common TP53-mutant allele in the initiation and maintenance of AML, a well-characterized liquid tumor where p53 loss is linked to aggressive and chemoresistant disease and one in which the potential GOF effects of mutant p53 have not been previously examined. We show that this p53 mutant accelerates leukemia onset and, strikingly, is required for leukemia maintenance, such that suppression of mutant p53 eliminates leukemia-initiating potential.
The biological basis of AML involves the emergence of leukemic blasts that have acquired mutations that drive increased proliferation of stem and progenitor cells, block their maturation, and promote their aberrant self-renewal (53, 54). In our models, the murine Trp53R172H mutant contributes to the latter activity, such that its elimination in leukemic cells releases the differentiation block. Similar results are observed with human AML cells harboring the equivalent R175H allele and likely explain the apparent requirement of certain other leukemia cells to additional mutant TP53 alleles (e.g., R248Q; see Supplementary Fig. S1B; ref. 28). Taking advantage of well-validated assays of self-renewal in the hematopoietic system, we show that p53R172H acts in nontransformed hematopoietic cells to promote self-renewal to a substantially greater degree than p53 loss, and that suppression of mutant p53 reverses these effects. Thus, in AML, mutant p53 can produce phenotypes analogous to bona fide oncogenes, such as the MLL–AF9 fusion oncoprotein, which also contributes to leukemia by enforcing aberrant self-renewal and exerts a continuous dependency for disease maintenance (55–57).
Although most TP53 mutations disrupt its DNA-binding capabilities, the resulting proteins are frequently stable and retain an intact transcriptional activation domain (4, 30). Some studies suggest that mutant p53 can be recruited to chromatin to induce gene expression via interaction with other transcriptional regulators, such as NF-Y and ETS2 (30, 58–60). Other studies support a model where mutant p53 proteins can directly bind to and perturb the function of other transcription factors, including p63, p73, and SREBP (13, 14, 58–60). In the AML system studied herein, mutant p53 increases the expression of the FOXH1 transcription factor, leading to its significant upregulation compared with Trp53Δ/Δ leukemias. Functional studies revealed that p53R172H is necessary and sufficient to induce FOXH1 which, in turn, binds stem cell–associated genes and contributes to enhanced self-renewal. The precise details of how mutant p53 induces FOXH1 remain to be determined and, to date, we have been unable to detect a robust ChIP signal for mutant p53 on DNA or validate an interaction with other p53 family members. Nevertheless, the importance of this molecular axis is supported by the conserved ability of mutant p53 to increase FOXH1 expression across species and by the significant upregulation of FOXH1 expression in CK-AML, a leukemia subtype characterized by a high frequency of TP53 mutations.
FOXH1 is expressed in embryonic stem cells and during early embryonic development. There, it is induced by the TGFβ family member Nodal (61) and acts to promote an EMT that takes place during gastrulation (43, 62). FOXH1 is not normally expressed in the adult hematopoietic system and, as such, mutant p53 appears to hijack FOXH1-driven programs to promote leukemia by enforcing the expression of genes normally involved in promoting stemness and cell plasticity during hematopoiesis. Like the Trp53R172H allele, p53 deficiency, as produced by two homozygous null Trp53 alleles, can also accelerate AML development by promoting aberrant self-renewal and enhancing leukemia-initiating potential (19). However, the aberrant program produced by the p53-null state is not as potent as that produced by p53R172H and does not depend on elevated levels of FOXH1. Presumably, this distinct rewiring of self-renewal programs explains why cells differentiate and lose leukemia-initiating capacity upon mutant p53 elimination to produce a p53-null state, the latter event being known to promote leukemogenesis if present as an initiating event. Thus, in this context, mutant p53 GOF produces a more profound phenotype than p53 loss while concomitantly creating unique molecular dependencies.
Our results may also have relevance to the pathogenesis of human AML. Specifically, beyond the GOF effect of p53R172H in leukemogenesis, this work reveals that mutant p53 can exert its phenotypes even prior to leukemia onset, leading to the continuous self-renewal of HSPCs in vitro and in vivo. Most previous GOF studies of mutant p53 have been in the context of established cancer and, to our knowledge, this is the first study to reveal that a GOF activity can produce molecular dependencies prior to neoplastic transformation. The effect is such that elimination of the mutant protein in premalignant cells promotes differentiation, impairs colony-forming ability, and reduces competitive fitness during long-term hematopoiesis in vivo. Although TP53 mutations are considered a later event during the progression of many solid tumors (63), they are present as “truncal” mutations with a high variant allele frequency in human AML such that we identified 35 AML cases with the R175H mutation and a median variant allele frequency (VAF) of 36% that showed evidence of clonal or subclonal presentation (64). Indeed, TP53 mutations are often observed in clonal hematopoiesis, a phenomenon where stem cells harboring leukemia-associated mutations expand in the peripheral blood of individuals in more than 0.02 VAF without overt disease (65–67). We identified 9 cases of clonal hematopoiesis that harbored TP53R175H mutations with a high VAF that ranged from 2% to 9%. Strikingly, 3 of 9 cases (∼33%) harbored only a single mutation, TP53 R175H, strongly suggesting that this is an early event. Of the remaining 6 cases, 2 individuals harbored clonal TP53R175H mutations whereas the remaining 4 contained subclonal mutations. Furthermore, individuals presenting with clonal hematopoiesis involving a TP53 mutation have the highest risk for progressing to AML (66). Taken together, these observations strongly suggest that TP53R175H mutation is an early, “first-hit” event in the pathogenesis of human AML.
Our data predict that some cancers might depend on continuous expression of mutant TP53 and are consistent with studies on the p53R248Q-mutant protein in mouse models of lymphoma, colon cancer, and others (68, 69). Nevertheless, it seems unlikely that all mutant TP53 alleles have GOF activity, and even those that do may impinge on distinct molecular mechanisms depending on context. For example, although p53R172H can display GOF effects by promoting invasion and metastasis in a pancreatic cancer model, it is not required for the sustained proliferation of pancreatic cancer cells, at least in vitro (13). Moreover, a recent report analyzing broad functional genomic datasets concluded that mutant TP53 was generally dispensable for the proliferation of human cell lines in culture (52). However, this study assessed only the effects of gene disruption on proliferation under in vitro conditions and did not examine signals associated with allele specificity or cellular context that are highly prevalent in the biology of p53 (8), whereas studies in vivo have confirmed a greater advantage of some of the hotspot mutations (70). Indeed, our functional experiments show a requirement for p53R175H in the leukemia-initiating potential of a human AML line, which could have been missed by performing a different type of analysis. In addition, upon detailed inspection of publicly available CRISPR screening data, we note that some TP53-mutant lines appear to depend on mutant TP53 for their survival (Supplementary Fig. S1B; ref. 30). Dissecting the underlying mechanism of this context specificity will be important to determine when and where mutant p53 proteins and their associated molecular programs may represent therapeutic targets.
Still, despite the perplexing range of molecular and biological mechanisms that have been attributed to GOF TP53 mutations, our studies reveal convergent features between mutant p53 GOF action in solid and liquid tumors, as well as in experimental models of induced pluripotency (44). In solid tumors, the best characterized phenotype associated with TP53 GOF mutations involves their ability to drive invasion and metastasis (12, 13, 69, 71, 72), which is often associated with an EMT and cellular plasticity that enables the acquisition of a more stem cell–like state. Consistent with this, the transcriptional profiles of p53-mutant tumors are enriched for gene signatures expressed in embryonic stem cells (73–75). In AML, Trp53R172H promotes self-renewal, as well as a transcriptional landscape enriched for gene signatures linked to stemness and, intriguingly, EMT. One such gene is Foxh1, which is known to promote EMT during embryonic development, yet we demonstrate here that it can drive aberrant self-renewal in AML. Remarkably, these same players contribute to the generation of iPS cells by the Yamanaka factors: Trp53R172H enhances iPS cell formation beyond p53 loss (16), and FOXH1 cooperates with p53 deficiency to achieve a similar effect (44). Collectively, these observations point to a convergent action of p53 GOF on cellular plasticity and stemness, which can influence distinct cancer processes depending on context.
Methods
Animal Models
All mouse experiments were approved by the Institutional Animal Care and Use Committee at Memorial Sloan Kettering Cancer Center (MSKCC). Trp53LSLR172H and Trp53LoxP mice were described previously (12). C57BL/6 (B6.SJL-Ptprca Pepcb/BoyJ (CD45.1) mice, thymectomized mice, and Vav1-Cre and Mx1-Cre mice were purchased from Jackson Laboratory and bred in a mouse facility at MSKCC.
Primary Cell Culture
Total BM cells from Trp53R172H/F;Vav1Cre, Trp53F/F;Vav1Cre and Trp53F/F (WT) mice (6–8 weeks) or purified BM c-KIT+ cells were transduced as described below with sh.Ren or sh.Trp53 retrovirus and plated in methylcellulose medium (Methocult M3434, StemCell Technologies). Cells were seeded at 10,000 total BM cells or 4,000 purified c-KIT+ cells per replicate. Colony-forming units were enumerated using a Zeiss Axio Observer microscope, and cells were replated (4,000 cells/replicate) every 7 to 10 days. Primary mouse PMN leukemias derived from Trp53R172H/F;Vav1-Cre and Trp53F/F;Vav1-Cre mice were cultured in 1:1 Dulbecco's Modified Eagle Medium (DMEM) to Iscove's Modified Dulbecco's Medium with 10% FBS (Gibco), 100 U/mL penicillin, 100 μg/mL streptomycin, 50 μmol/L 2-mercaptoethanol, and recombinant mouse stem cell factor (SCF; 50 ng/mL), IL3 (10 ng/mL), and IL6 (10 ng/mL) at 37°C in 5% CO2. Puromycin and doxycycline were used at a final concentration of 1 μg/mL for all cell culture assays.
Cell Lines
The human leukemia line KY821 (JCRB cell bank) was grown in RPMI-1640 and 20% FBS (Gibco). KL922 (Trp53Δ/Δ), KL973 (Trp53R172H/11B3), and KL974 (Trp53R172H/Δ) primary mouse cell lines (19, 25) were cultured with RPMI, 10% FBS (Gibco), and recombinant mouse SCF (50 ng/mL), IL3 (10 ng/mL), and IL6 (10 ng/mL). PlatinumE (76) cells were a gift from Craig Thomson's laboratory and HEK293T were purchased from ATCC (CRL-3216) and were used for retroviral production while maintained in DMEM with 10% FBS. Media supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and cultures were incubated at 37°C, 5% CO2. All cells tested negative for Mycoplasma (MP0025-1KT) and were not maintained for longer than 10 passages in culture. For cell line authentication, we confirmed p53 expression by Western blot analysis, and Sanger sequencing confirmed the specific mutation.
Xenotransplantation
Experiments were carried out in accordance with institutional guidelines approved by MSKCC. Immunodeficient NSG mice (6–10 weeks old) were bred under pathogen-free conditions and were sublethally irradiated (200–250 cGy) 3 to 24 hours before transplantation. KY821 cells were injected by tail-vein injection. Complete blood count analysis was performed on peripheral blood collected from submandibular bleeding using a Drew Hemavet Analyzer (Veterinary Diagnostics).
Histology and Microscopy
Tissues were dissected from mice for fixation overnight in 10% formalin (Thermo Fisher Scientific). Bones were decalcified as per the manufacturer's instructions (IDEXX BioResearch), and fixed tissues were dehydrated and embedded in paraffin for sectioning. Paraffin sections (5 μm) were prepared and stained with hematoxylin and eosin (Leica Autostainer XL).
Flow Cytometry
Single-cell suspensions were prepared from BM, spleen, or peripheral blood. Red blood cells were lysed with ammonium–chloride–potassium (ACK) buffer, and the remaining cells were resuspended in PBS with 3% FBS. Nonspecific antibody binding was blocked by incubation with 20 μg/mL rat IgG (Sigma-Aldrich) for 15 minutes, and cells were then incubated with the indicated primary antibodies for 30 minutes on ice. Stained cells were quantified using a Fortessa analyzer (BD Biosciences) or isolated with a FACSAria II (BD Biosciences). FlowJo software (TreeStar) was used to generate flow cytometry plots. More details on FACS antibodies can be found in Supplementary Table S2.
BM Competitive Transplantation
Femurs and tibiae were isolated from donor Trp53F/F (WT), Trp53F/F;Mx1-Cre or Trp53R172H/F;Mx1-Cre (CD45.2), as well as WT (CD45.1) mice. Bones were flushed with PBS and the single-cell suspension was centrifuged (5 minutes, 0.5 × g, 4°C) and treated with red cell ACK lysis buffer, as described above. Total nucleated BM cells were resuspended in PBS, passed through a 40-μm cell strainer, and counted. Donor cells (1 × 106 per genotype, per mouse) were mixed 1:1 with support BM cells (CD45.1) and transplanted via retro-orbital injection into lethally irradiated (950 Rad) CD45.1 recipient mice. Chimerism was monitored by flow cytometry (anti-CD45.1 and anti-CD45.2; BD Biosciences) of peripheral blood at 4-week intervals posttransplant three weeks after last pIpC injection and for 16 weeks, at which time mice were sacrificed and chimerism was assessed in other hematopoietic compartments (BM).
Retroviral Transduction
HSPCs were isolated by sorting Lin−c-KIT+Sca1+ cells or enriched using an autoMACS Pro separator (Miltenyi Biotec) using CD117 Magnetic Microbeads (Miltenyi Biotec, 130-091-224), then cultured in the presence of 50 ng/mL SCF, 10 ng/mL IL3, and 10 ng/mL IL6, and infected with concentrated supernatants of LMNe-sh.Ren/sh.p53-BFP or MLPe sh.Ren/sh.p53/sh.FoxH1 retrovirus after 24 and 48 hours (more details about the shRNAs in Supplementary Table S2). Transduction efficiency was determined by reporter fluorescence at 48 hours, and 5,000 sorted cells were injected into CD45.1 mice as described above.
Cell Viability and DNA Synthesis
Apoptosis assays were performed by staining with Annexin V (BD Pharmingen) according to the manufacturer's instructions, in combination with DAPI. For cell-cycle studies, BrdU (BD Pharmingen) was used per manufacturer's instructions and stained with 2 μg/mL DAPI prior to flow cytometric analysis.
Quantitative RNA Expression Assays
Total RNA was extracted from cells using the RNeasy Plus Mini Kit (Qiagen). RNA quantity and quality were determined using an Agilent 2100 Bioanalyzer. RNA-seq libraries were prepared from total RNA by polyA selection using oligo-dT beads (Life Technologies) according to the manufacturer's instructions. The resulting poly-A+ RNA served as input for library construction using standard Illumina protocols. Sequencing was performed on an Illumina HiSeq 2500 sequencer using 50 bp pair-end reads. For mRNA quantification, total RNA was used for cDNA synthesis (Agilent). Real-time PCR reactions were carried out using SYBR Green Master Mix (QuantaBio) and a Viia7 (Applied Biosystems).
RNA-seq Analysis
RNA-seq library construction and sequencing were performed at the integrated genomics operation (IGO) Core at MSKCC. Poly-A selection was performed. For sequencing, approximately 30 million 50-bp paired-end reads were acquired per replicate condition. Resulting RNA-seq data were analyzed by removing adaptor sequences using Trimmomatic (77). RNA-seq reads were then aligned to GRCh37.75 (hg19) with STAR (78), and genome-wide transcript counting was performed by HTSeq (79) to generate a matrix of fragments per kilobase of exon per million fragments mapped. Gene expressions of RNA-seq data were managed by hierarchical clustering based on one minus Pearson correlation test using Morpheus (https://software.broadinstitute.org/morpheus/). All results from the RNA-seq experiments can be found under GSE125097.
GSEA
GSEA was performed using gene set as permutation type, 1,000 permutations, and log2 ratio of classes, or with gene set and Signal2Noise as metrics for ranking genes. Gene sets used in this study were identified from the Molecular Signatures Database (MSigDB Curated v4.0). Gene pathways and functions were assessed using Ingenuity Pathway Analysis (Qiagen Bioinformatics).
Expression of Gene Signatures in Human AML and Normal Cells
Visualization of gene signatures were derived from transcriptional data as log2 expression values for different karyotypes of patients with AML described previously in GSE42519 (80). For normal hematopoietic cells, values were averaged over all genes in each signature and visualized by using Sinaplot. Student t test was performed using the R programming language (https://www.R-project.org/).
Quantification and Statistical Analysis
All P values were calculated using unpaired two-tailed Student t test with GraphPad Prism software, unless otherwise described in the methods or figure legends. No specific randomization or blinding protocol was used for these analyses. Statistically significant differences are indicated with asterisks in figures with the accompanying P values in the legend. Error bars in figures indicate SD or SEM for the number of replicates, as indicated in the figure legend.
Plasmids and Viral Production
All vectors were derived from the murine stem cell virus (MSCV; Clontech) retroviral vector backbone. miRE-based shRNAs were designed and cloned as described previously (27) into the LT3GEPIR (TRE3G-GFP-miRE-PGK-PuroR-IRES-rtTA3) vector (81). The mouse Foxh1 cDNA was cloned in a pVXL-hygromycin-mFoxh1 vector (provided by Yilong Zou, Massagué lab, MSKCC). All constructs were verified by sequencing. Lentiviruses were produced by cotransfection of 293T cells with 10 μg LT3GEPIR construct and helper vectors (3.75 μg psPAX2 and 1.25 μgVSV-G). For retroviral infection, PlatinumE cells were plated in 15-cm diameter dishes each transfected with 54 μg of MSCV vectors and 2.7 μg of VSV-G and 8.1 μg of pCl-eco. Transfection of packaging cells was performed using HEPES-buffered saline and CaCl2 or Mirus transfection reagent (MIR 2304). Viral supernatants were passed through a 0.45-μm filter (Millipore) and concentrated with Centrifugal Filter units (Millipore) to obtain high virus titer. Concentrated virus was supplemented with 8 μg/mL of polybrene (Sigma) before adding to target cells.
Immunoblotting
For protein lysates, cells were incubated with RIPA buffer supplemented with protease inhibitors (Protease inhibitor tablets, Roche) for 30 minutes and cleared by centrifugation (15 minutes 14.000 rpms 4C). Protein was quantified using the DC protein assay (Bio-Rad). The following antibodies were used for immunoblotting: β-Actin (ac-15, Sigma), mp53 (CM5, Leica Microsystems), hp53 (DO-1, Santa Cruz Biotechnology), mFOXH1 (Abcam 49133), and hFOXH1 (Abcam 102590).
ChIP
ChIP was performed as described previously (82). Briefly, KL974 (Trp53R172H/Δ) cells were fixed with 1% formaldehyde for 15 minutes, and the cross-linking reaction was stopped by adding 125 mmol/L glycine. Cells were washed twice with cold PBS and lysed in buffer (150 mmol/L NaCl, 1%v/v Nonidet P-40, 0.5% w/v deoxycholate, 0.1% w/v SDS, 50 mmol/L Tris pH 8, 5 mmol/L EDTA) supplemented with protease inhibitors. Cell lysates were sonicated using a Covaris E220 Sonicator to generate fragments less than 400 bp. Sonicated lysates were centrifuged and incubated overnight at 4°C with specific antibodies (FOXH1 Abcam 49133). Immunocomplexes were recovered by incubation with 30 μL protein A/G magnetic beads (Thermo Fisher Scientific) for 2 hours at 4°C. Beads were sequentially washed twice with RIPA buffer, increasing stringency ChIP wash buffers (150 mmol/L NaCl, 250 mmol/L NaCl, 250 mmol/L LiCl), and finally TE buffer. Immunocomplexes were eluted using elution buffer (1% SDS, 100 mmol/L NaHCO3), and cross-linking was reverted by the addition of 300 mmol/L NaCl and incubation at 65°C overnight. DNA was purified using a PCR Purification Kit (Qiagen). Input chromatin was used for estimation of relative enrichment. The results for the ChIP-seq experiments can be found under GSE125097.
ChIP-seq Library Preparation, Illumina Data Analysis, and Peak Detection
ChIP-seq libraries were prepared at the Center for Epigenetic Research (MSKCC) using the NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina (New England BioLabs) following the manufacturer's instructions. Reads were trimmed for quality and Illumina adapter sequences using “trim_galore” before aligning to mouse assembly mm9 with bowtie2 using the default parameters. Aligned reads with the same start site and orientation were removed using the Picard tool MarkDuplicates (http://broadinstitute.github.io/picard/). Density profiles were created by extending each read to the average library fragment size and then computing density using the BEDTools suite (http://bedtools.readthedocs.io). Enriched regions were discovered using MACS2 and scored against matched input libraries (fold change > 2 and P < 0.005). Dynamic regions between two conditions were discovered using a similar method, with the second ChIP library replacing input. Peaks were then filtered against genomic “blacklisted” regions (http://mitra.stanford.edu/kundaje/akundaje/release/blacklists/mm9-mouse/mm9blacklist.bed.gz) and those within 500 bp were merged. All genome browser tracks and read density tables were normalized to a sequencing depth of ten million mapped reads. Dynamic peaks were annotated using linear genomic distance, and motif signatures were obtained using the “de novo” approach with Homer v4.5 (http://homer.ucsd.edu/).
Disclosure of Potential Conflicts of Interest
S.W. Lowe has ownership interest (including stock, patents, etc.) in PMV Pharmaceuticals. No potential conflicts of interest were disclosed by the other authors.
Authors' Contributions
Conception and design: E. Loizou, S.W. Lowe
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): E. Loizou, A. Banito, A. Mayle
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): E. Loizou, A. Banito, G. Livshits, Y.-J. Ho, R.P. Koche, C.-C. Chen, S. Kinalis, F.O. Bagger, E.R. Kastenhuber, B.H. Durham
Writing, review, and/or revision of the manuscript: E. Loizou, A. Banito, G. Livshits, F.J. Sanchez-Rivera, A. Mayle, E.R. Kastenhuber, B.H. Durham, S.W. Lowe
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Y.-J. Ho
Study supervision: S.W. Lowe
Acknowledgments
The authors are grateful to Charles J. Sherr for extensive scientific discussion, suggestions for experiments, and help in formulating and editing the manuscript. We thank Omar Abdel-Wahab, Ross L. Levine, and Scott Armstrong for their key input and reagents, and Elli Papaemmanuil for providing analysis of human AML datasets. We also thank members of the Lowe laboratory for comments and discussions, Yilong Zou and Qiong Wang for providing us with the mFOXH1-expressing vector, and Jiajun Zhu for helping to optimize ChIP-seq conditions. This work was supported by a grant from the NCI (R01 CA190261) and the Memorial Sloan Kettering Cancer Center Support Grant (P30 CA008748). E. Loizou was supported by the Dorris J. Hutchison Student Fellowship (SKD). A. Banito was supported by a European Molecular Biology Organization long-term fellowship. E.R. Kastenhuber was supported by an F31 National Research Service Award predoctoral fellowship from the NCI/NIH under award number F31CA192835. F.J. Sanchez-Rivera is a Howard Hughes Medical Institute Hanna Gray Fellow and was partially supported by an MSKCC Translational Research Oncology Training Fellowship (NIH T32-CA160001). S.W. Lowe is an investigator at the Howard Hughes Medical Institute and is the Geoffrey Beene Chair of Cancer Biology.