Abstract
Transcriptional regulators, including the cohesin complex member STAG2, are recurrently mutated in cancer. The role of STAG2 in gene regulation, hematopoiesis, and tumor suppression remains unresolved. We show Stag2 deletion in hematopoietic stem/progenitor cells (HSPC) results in altered hematopoietic function, increased self-renewal, and impaired differentiation. ChIP-sequencing revealed that while Stag2 and Stag1 can bind the same loci, a component of Stag2 binding sites are unoccupied by Stag1 even in Stag2-deficient HSPCs. While concurrent loss of Stag2 and Stag1 abrogated hematopoiesis, Stag2 loss alone decreased chromatin accessibility and transcription of lineage-specification genes, including Ebf1 and Pax5, leading to blunted HSPC commitment to the B-cell lineage. Our data illustrate a role for Stag2 in transformation and transcriptional dysregulation distinct from its shared role with Stag1 in chromosomal segregation.
One Sentence Summary Stag1 rescues topologically associated domains in the absence of Stag2, but cannot restore chromatin architecture required for hematopoietic lineage commitment
Main text
Cell-type specific transcriptional programs are regulated in part by the activity of tissue-specific transcription factors and enhancer elements within structurally defined topologically associating domains (TADs) (1-3). The genes which contribute to transcriptional regulation, including members of the cohesin complex, are frequently mutated in human cancers, including leukemias(4-9). Cohesin is a tripartite ring comprised of three structural proteins SMC1A, SMC3, and RAD21; this core complex binds to either STAG1 or STAG2. STAG2, which is an X-linked gene, is the most commonly mutated cohesin complex member in human cancer, with recurrent nonsense, frameshift, and missense mutations in Ewing’s Sarcoma (40-60%)(9), bladder cancer (20-35%)(7, 8), glioblastoma (4-5%)(7), myelodysplastic syndrome (MDS)(5-20%)(5, 6), and acute myeloid leukemia (AML)(2-12%)(5). This is in contrast to SMC1A, SMC3, and RAD21, which are more commonly mutated in myeloid malignancies than in a broad spectrum of human cancers. The cohesin complex is essential in pleiotropic cellular and gene regulatory functions, including chromosome segregation(10, 11) and in the formation of DNA loops(3, 12-14) which regulate gene expression(13, 15, 16). However, the high mutational frequency and unique mutational spectrum of STAG2 in human cancers(5-7) suggests a distinct role in homeostasis and tumor suppression(17, 18) which has not been delineated.
Results
Loss of Stag2 alters hematopoietic stem cell function
In order to elucidate the role of Stag1 and Stag2 in hematopoietic function/transformation, we generated Stag1 and Stag2 conditional knockout alleles. We induced somatic deletion of Stag1 or Stag2 in hematopoietic cells through Mx1-Cre mediated excision (Figure S1A-H). Following polyinosinic:polycytadinic (PIPC)-mediated deletion, Mx1-cre Stag2 −/− mice compared to cre-negative Stag2fl/fl (referred henceforth as KO and WT respectively) manifested an increase in hematopoietic stem cells (HSC), including a significant (2.2-fold; p<0.01) increase in the proportion of LSK (Lineage−Sca1+cKit+) cells (Figure 1A). Stag2 loss, but not Stag1 loss, induced an expansion of multipotent progenitors (MPP: LSK+Cd150−Cd48+Cd127−; p<0.04), short term HSCs (ST-HSC: LSK+Cd150−Cd48−; p<0.001), and long-term HSCs (LT-HSC: LSK+Cd150+Cd48−; p<0.02) (Figure S2A). We also observed a significant increase in granulocyte macrophage progenitors (GMP: Lineage−cKit+Sca1−Cd34+Fcγ+; p<0.001; Figure S2B-C) in the Stag2-deficient hematopoietic compartment.
We next investigated the role of Stag2 and Stag1 in hematopoietic self-renewal. Stag2-deficient hematopoietic cells displayed increased serial replating capacity in methylcellulose culture assays (≥7 platings), whereas WT and Stag1 KO cells were not able to form colonies beyond 3 platings (Figure 1B). We next performed competitive transplantation assays to assess the impact of Stag2/Stag1 loss on self-renewal in vivo. We observed reduced Stag2-deficient derived chimerism in the peripheral blood of mice reconstituted with Stag2 KO and WT competitor cells (p<0.001; Figure S2D-F). However, within the bone marrow of transplant recipient mice, we found a significant increase in Stag2 KO derived chimerism, which was most significant in the LSK compartment (p<0.001) including in LT-HSCs (p<0.001; Figure 1C) but not in lineage positive cells (p≤0.37). Stag1 loss did not impact in vivo self-renewal or differentiation output (Figure 1D; S2G-I). These data suggest that Stag2 loss induces both an increase in self-renewal and reduced differentiation capacity in HSCs, which are critical features of hematopoietic transformation.
To determine if Stag2 loss was associated with the expansion of hematopoietic cells with clonal cytogenetic alterations, we performed karyotype analysis in Stag2 and Stag1 WT and KO bone marrow cells. Chromosomal aberrations by metaphase karyotyping were not detected in either Stag2- or Stag1-deficient hematopoietic cells (n=7; Figure S3A-B). We did observe an increase in the proportion of cells with premature sister chromatid separation in Stag2 KO bone marrow (Figure S3C); however, consistent with metaphase cytogenetics, we did not observe clonal copy number alterations on low-pass whole genome sequencing (Figure S3D-E). This data suggested that the increase in hematopoietic self-renewal induced by Stag2 loss is not due to genomic instability and resultant clonal evolution.
Stag2 and Stag1 co-deletion is synthetic lethal
The observation that Stag2-deficient HSCs displayed increased self-renewal is in contrast to the lethality observed with loss of the core cohesin component Smc3 in hematopoietic cells(19). We next sought to evaluate potential compensatory function among the Stag paralogs. We found Stag1 transcript and protein expression was significantly increased in Stag2 KO hematopoietic cells, whereas Stag2 expression was not altered in Stag1 KO hematopoietic cells (Figure S4A-D). Analysis of the AML TCGA dataset showed that AML patients with STAG2 mutations expressed higher levels of STAG1 (p<0.006; Figure S4E), suggesting compensatory regulation. We hypothesized that Stag1 and Stag2 may have redundant roles with respect to chromatid segregation such that Stag2 loss does not fully abrogate cohesin function due to residual Stag1 function. In vitro studies have recently nominated STAG1 as a synthetic lethal target in STAG2 mutant cell lines(20); however, this has not been assessed in vivo. We generated mice with hematopoietic cells retaining one or zero copies of Stag1 on the background of a Stag2 (hemizygous male) KO genotype. Stag2-/y/Stag1+/- mice developed rapid, progressive thrombocytopenia (p<0.001) and impaired survival (p<0.001) due to bone marrow failure (median survival of 27.7 weeks). Complete loss of Stag1 and Stag2 in hematopoietic cells induced rapid lethality (median survival 0.7 weeks; Figure 1E; Figure S4F). Stag1/Stag2 deficient mice were characterized by pancytopenia and bone marrow aplasia, consistent with redundancy in hematopoietic function and in alignment with the absolute requirement for core cohesin complex members (Figure 1F). Similar to mice with bi-allelic deletion of Smc3, we observed marked chromosomal alterations on metaphase spreads of the Stag1/Stag2 deleted bone marrow (Figure S4G). Collectively, these data suggest a role for Stag2 in gene regulation and tumor suppression that is not shared with Stag1, whereas they have a shared, redundant role in chromosomal segregation.
Stag2 alters transcriptional lineage commitment
We hypothesized that Stag2 loss induced alterations in transcriptional output which dysregulate hematopoietic function and promote transformation. To investigate transcriptional mechanisms regulating self-renewal and differentiation in the context of Stag2 loss, we performed RNA-sequencing on Stag2 KO and WT LSK cells. We identified 186 genes which were significantly downregulated and 42 genes which were significantly upregulated in Stag2 KO compared to WT cells (Figure S5A). We observed reduced expression of Ebf1, Vbrep3, Cd19, Bank1, and Pax5, suggesting a loss of B-cell lineage commitment(21, 22) in Stag2-deficient stem/progenitor cells. We observed reduced expression of Vwf(23), Hba-a1, Hba-a2, Hbb-b1(24), Fcgr2b(25), Ccr2(26), Spib(27), S100a8, and S100a9(28), genes associated with myeloid/erythroid lineage commitment. Gene set enrichment analysis (GSEA) revealed that Stag2 deficient LSKs showed negative enrichment of genes associated with cell lineage specification (Figure S5B). We further found increased expression of the Hoxb4-target Hemgn(29), as well as of Itgb3(30), a Periostin-binding integrin shown to mediate HSC self-renewal. Coupled with downregulation of Flt3(31) and Selp(32) (P-selectin), these findings indicate an LSK pool that is transcriptionally skewed towards increased self-renewal.
Given the heterogeneity of the LSK compartment, we sought to determine if the transcriptional alterations observed in bulk LSKs were due to a population wide shift in expression or a result of differential expression within a subset of LSKs. We performed single cell RNA-sequencing (scRNAseq) of ∼24,000 Lin− hematopoietic stem/progenitor cells (HSPC) from Stag2 KO (n=3) and WT (n=3) mice (cohort annotations in Figure S5C-E). We found that within the HSC population, representing ∼8% of cells, that the compartment distribution was skewed with KO HSCs having enriched expression of cell cycle gene signatures (p<10−63; Figure 1G, S5F top panel). Concomitantly, these HSC showed decreased expression of early lymphoid commitment signatures (p<10−14; Figure 1G, S5F bottom panel) even at the earliest HSC states, consistent with negative enrichment for cell specification. We expanded upon these findings by comparing bulk LSK differentially expressed genes to gene expression data from dormant and active HSCs separately isolated through genetic lineage tracing studies(33). We observed that genes that were decreased in expression in Stag2-deficient mice were most highly expressed in slow-cycling quiescent HSCs, while the upregulated genes were most highly expressed in the proliferative MPP1 fraction (LSK, Cd34+, Flt3−) (Figure S5G). Similar findings were observed when comparing our data to single cell RNA-seq data, such that the most downregulated genes in Stag2-deficient LSKs were most highly expressed in the most quiescent HSCs (Figure S5H). Collectively, these analyses highlight a transcriptional signature associated with decreased lineage commitment, decreased quiescence, and enhanced self-renewal in the setting of STAG2 loss.
Stag2 loss impairs differentiation and induces dysplasia
To determine the consequences of these transcriptional alterations in vivo, we assessed the phenotype of mice with Stag2-deficient hematopoiesis over time. Hematopoietic-specific Stag2 loss resulted in progressive leukopenia (p<0.02) and thrombocytopenia (p<0.01; Figure S6A). We observed an expansion in myeloid cells and a significant reduction in B lymphocytes (p<0.001; Figure 2A) in the peripheral blood, which was not seen with Stag1 deletion. Stag2 loss, but not Stag1 loss, induced morphologic alterations including an increase in immature myeloid cells, an expansion of small hypolobated megakaryocytes, and nuclear:cytoplasmic dyssynchrony in the erythroid lineage (Figure 2B). These abnormalities are features of human myelodysplasia—consistent with the observation that 5-15% of MDS patients have loss-of-function mutations in STAG2(5, 6).
To determine at which level of differentiation multi-lineage dysplasia emerges, we analyzed lineage composition through analysis of scRNAseq data (Figure 2C-D). Supporting the reduction in B cell output observed in the peripheral blood, analysis of scRNAseq data suggested a significant decrease in B cells and in B cell progenitors (Figure 2E). Although the paucity of lymphoid cells precluded further transcriptional tracing, we observed population shifts in erythroid and granulocyte development consistent with observed histologic myelodysplasia. Flow cytometric analysis of erythroblast maturation revealed attenuation at Stage III of erythropoiesis(34) (Figure 2F, S6B-C), a stage that is demarcated by condensed cell size as measured by forward scatter (FSC) and decreased CD44 expression relative to prior stages of erythroid differentiation. Stag2-deficient erythroid cells were also characterized by morphologic dysplasia; immunohistochemical analysis demonstrated a marked decrease in the proportion of Ter-119+ erythroid cells (Figure 2G). By comparison, Stag1 KO mice did not manifest hematopoietic alterations, morphologic abnormalities, or defects in erythroid differentiation (Figure S6A-C). Transcriptional analysis of the erythroid lineage in Stag2 deficient cells further supported impaired terminal maturation (p<10−44; Figure 2H). Genes defining this principle component define the progression from common myeloid progenitor to megakaryocyte erythroid progenitor to erythroid A (Figure S6D-E). Overall, the most significant transcriptional difference identified in the scRNAseq was impaired granulocytic terminal maturation (p<10−92; Figure 2I). Genes defining this principle component define the progression from common myeloid progenitor to granulocyte macrophage progenitor to granulocyte (Figure S6F-G).
Given the alterations in transcriptional output and differentiation potential, we hypothesized that Stag2 loss led to altered chromatin accessibility at differentially expressed loci. We therefore performed RNA sequencing and ATAC-sequencing on sorted Stag2 WT and KO GMP and erythroid progenitor (CFU-E: Lineage−cKit+Sca1−Cd34−Fcγ−Cd105+) cells. Compared to WT GMPs, Stag2-deficient GMPs had enrichment for a stem-cell gene expression signature associated with increased Evi1 expression and negative enrichment of myeloid cell development signatures by GSEA (Figure 2J). Similarly, Stag2-deficient CFU-Es had increased expression of genes normally expressed in HSCs and in the megakaryocyte lineage, consistent with the increased self-renewal and impaired differentiation seen with Stag2 loss (Figure 2K). Integrated analysis of RNA-seq and ATAC-seq revealed that a significant subset of the downregulated genes also had reduced chromatin accessibility. Both GMPs and CFU-E shared loss of accessibility and expression for genes with either CTCF/CTCFL (BORIS) or PU.1(ETS) motifs (Figure 2L-M). CTCF is a well-described cohesin binding partner(35) that cooperates with cohesin to mediate enhancer/promoter associations(12). Notably, PU.1 and its target genes have a critical role in determining hematopoietic lineage commitment(36, 37).
Stag1/2 possess shared and independent chromatin binding
It has been shown using in vitro model systems that cohesin is essential for genomic cis-interactions and that rapid depletion of cohesin ring members result in complete loss of interphase architecture(3, 14, 38). However, these model systems do not reflect a physiologic loss/partial loss of a cohesin complex member akin to the genomic alterations observed in either human cancers or the germline cohesinopathy, Cornelia de Lange syndrome(39). As the complete loss of cohesin structural loop components may not be compatible with a viable cell division event, the absence of transcriptional changes observed using in vitro systems of cohesin degradation(14) may not reflect the pathophysiology of Stag2 loss of function. We next sought to determine how Stag2-loss affected chromatin topology in primary cells using Hi-C chromosome conformation capture in hematopoietic progenitor cells. Stag2 KO cells did not show alterations in topologically associating domain (TAD) boundaries, in global insulation, and did not show differences in the spatially segregated genomic regions known as A/B compartments comparted to WT hematopoietic progenitor cells(40) (Figure 3A-C).
Given the lack of changes in global structure, we sought to determine if Stag2 loss would affect the ability of Ctcf of the remaining structural cohesin components Smc1a and Smc3 to localize to canonical cohesion/CTCF binding sites. Chromatin immunoprecipitation sequencing revealed that the occupancy of these three proteins was largely unaffected by the presence or absence of Stag2 in HSPCs (Figure S7A-C). We hypothesized that complete loss of Stag2 might not alter higher order DNA topology due to redundancy with Stag1 at CTCF bound sites. We therefore performed ChIP-seq to delineate Stag2 and Stag1 occupancy in HSPCs from Stag2 WT and KO mice. We identified a set of peaks where Stag2 and Stag1 were bound in WT mice and where Stag1 occupancy increased in the absence of Stag2 (n=2,493; Figure 3D, S7D-E). Conversely, we identified a second set of peaks where Stag2, but not Stag1, was bound in WT HSPCs and lost in Stag2 KO cells without compensatory Stag1 occupancy (n=10,809; Figure 3E, S7F-G). Previous work has demonstrated CTCF-dependent and independent localization of the cohesin complex(41, 42) which in epithelial cell lines have preferential binding sites for Stag1 and Stag2(43). We hypothesized that altered regulation of Stag2-bound loci in WT cells which are not bound by Stag1 in WT or KO cells, were key to the altered differentiation and self-renewal seen in Stag2 KO mice. We found that loci with both Stag2 and Stag1 binding had the highest enrichment for CTCF binding (Figure 3E-F).
Loss of chromatin insulation impairs transcriptional output
Stag1 was able to bind a subset of loci previously occupied by Stag2 or both Stag1 and Stag2 in WT cells; this included TAD boundaries determined by Hi-C (Figure 3F). By contrast, when we focused on Stag2-only bound sites, we observed locus-specific alterations in short-range interactions (as calculation by the insulation metric; Figure S6B). We quantified these alterations through insulation scores (IS) as a measure of cumulative local contacts and insulation score changes (ISC) that reflect local chromosomal organization changes. To examine whether loss of Stag2 binding affects local chromosomal organization, we compared ISC in WT and Stag2 KO HSPCs and identified 71,815 ISC bins that showed local chromosomal contact changes (10kb bins with insulation score change less than −0.1 or greater than 0.1, Figure S8A, left panel). We then overlapped ISC bins with Stag1/2 co-bound and Stag2-specific peaks. Annotation analysis of both Stag1/Stag2 common and Stag2-specific peaks showed enrichment of Stag2-specific ISC peaks within promoter regions (Figure S8A, right panel), suggesting that ISC seen in Stag2- deficient cells contribute to altered gene expression.
These data suggested that the role of Stag2 in gene regulation and in hematopoiesis might be due to regulation of loci bound by factors other than CTCF. To determine the impact of Stag2 loss on chromatin structure and transcriptional output we performed RNA-seq and ATAC-seq in WT and Stag2 KO HSPCs. Similar to gene expression/ATAC-seq data in sorted hematopoietic subsets, the majority of genes with significantly altered chromatin accessibility and gene expression in the absence of Stag2 had decreased accessibility and reduced transcription (Figure 4A). Several of these peaks were in proximity to critical B-cell and myeloid lineage defining transcription factors, including Ebf1, Pax5, and Cebpb. To uncover transcription factor networks underlying these changes, we performed motif enrichment on peaks with concomitant reduction in accessibility/gene expression. We observed enrichment for Gata, Ebf, and E2A targets (Figure 4B), but not for Cebpb motifs. The genes associated with loss of insulation by ISC significantly overlapped with those loci with decreased accessibility and decreased expression, with Ebf1 and Pax5 among the genes with the greatest magnitude of insulation change (Figure 4C, S8B-D). We therefore performed motif analysis on Stag1/2 common and Stag2-specific sites by dichotomizing those that were bound and not bound by CTCF. As expected, loci with shared Stag1/Stag2 occupancy and CTCF binding sites were enriched for CTCF and CTCFL (BORIS) binding motifs. By contrast, Stag2-specific loci (not bound by Stag1) were enriched for key lineage-specific transcription factor motifs including ERG, IRF8, and PU.1 (Figure 4D). These transcription factors are essential for myeloid and lymphoid development (44, 45) and attenuated function of these transcription factors has transformative potential in murine(37) and human hematopoietic cells(46-48).
Altered Ebf1 chromatin structure results in B-cell blockade
We next sought to determine if the altered expression of hematopoietic transcription factors was due to alterations in the population distribution in HSPC or was due to altered expression of these factors in purified hematopoietic populations, including in multipotent progenitors antecedent to full lineage commitment. We measured PU.1 and Ebf1 expression and B cell differentiation in MPPs and in common lymphoid progenitors (CLP: LSK+Cd150−Cd48+ Cd127+) from Stag2 KO and WT mice. We found that Ebf1 expression was reduced/absent in purified Stag2 KO MPPs (p<0.04, Log2FC=-1.8) and CLPs (not detectible in KO), while there was no significant difference in PU.1 expression in either cell type (Figure 4E). Consistent with these data, B cell maturation was blocked from the pro-B cell to early pre-B cell stage(49) in Stag2 KO mice, with almost no late pre-B cells (Figure 5A). These findings were further bolstered by in vitro colony assays in B cell cytokine enriched methylcellulose where Stag2 KO HSPCs failed to generate B cell colonies (Figure 5B). We next investigated if the same was true in Stag2-mutant MDS patients by enumerating B cells and B cell progenitors in STAG2-mutant MDS patient bone marrow samples compared to a cohort of non-MDS individuals with cytopenias (Figure 5C, S8E). Immature B cells (CD34+CD19+) were decreased by 1.7-fold (p<0.01) in STAG2-mutant MDS patients, demonstrating that altered B-cell lineage commitment is observed in STAG2-mutant MDS.
Given that PU.1 can regulate myeloid and lymphoid lineage commitment(37) and the enrichment for PU.1 motif/targets in our epigenomic/transcriptional analysis, we investigated the role of PU.1 in Stag2-deficient alterations in B-cell lineage commitment and self-renewal. PU.1 silencing reduced B-lineage output derived from WT hematopoietic cells, similar to the effects of Stag2 deletion (Figure 5D). However, the reduced B-cell lineage output of Stag2 KO cells was not further attenuated by PU.1 silencing as assessed by flow cytometric analysis of B220 expression and B-lineage colony formation (Figure 5D). By contrast, overexpression of PU.1 was not sufficient to restore B220 expression in B cell methylcellulose cultures (Figure 5D) or to attenuate the increased expression of the stem/progenitor marker cKit seen on Stag2-deficient hematopoietic cells (Figure 5E). Moreover, overexpression of PU.1 failed to attenuate the capacity of Stag2- deficient hematopoietic cells to serially replate (Figure S8F-H).
This led us to hypothesize that key transcription factors downstream of PU.1 which are bound by Stag have locus specific alterations in conformation in the absence of Stag2 that lead to alterations in target gene expression with functional import. Ebf1 has four Stag2 bound sites in hematopoietic progenitors, all of which are not bound by Stag1 in WT or KO cells (Figure 5F). As noted above, we observed near-complete abrogation of Ebf1 transcription from the onset of B-lineage commitment (Figure 4E). We observed a significant reduction in local insulation throughout the Ebf1 locus (Figure 5G) including loss of cis-interactions at three Stag2 binding sites (Figure 5H). Consistent with this hypothesis, expression of Ebf1 in Stag2 KO progenitors (Figure S8I) fully restored B-lineage marker expression and B-cell colony formation in vitro (Figure 5I). Moreover, Ebf1-expressing Stag2 KO progenitors had decreased cKit expression (Figure 5J) and Ebf1 restoration abrogated the serial replating capacity of Stag2-deficient cells (Figure 5K). Taken together, these data demonstrate a key role for Stag2-mediated regulation of Ebf1 expression in modulating the balance between hematopoietic differentiation and self-renewal.
Discussion
The cohesin complex is essential in pleiotropic cellular and gene regulatory functions, including in chromosome segregation(10, 11) and in maintaining DNA interactions(3, 14) which regulate gene expression(13, 15, 16). Here we demonstrate a non-redundant role of Stag2 and Stag1 in hematopoiesis, with a specific role for Stag2 in regulating the balance between hematopoietic differentiation and self-renewal. In addition, we show that Stag2 bound loci that are not bound by Stag1 undergo locus specific changes in local interactions that, in the absence of Stag2, leads to altered expression of key loci governing hematopoietic function. Taken together, these data illustrate a critical link between Stag2, DNA interactions, and gene regulation that contributes to hematopoietic transformation.
Stag2 and Stag1 have both shared and distinct roles(43). These paralogs have a redundant role in chromatid separation such the presence of either protein within the cohesin complex is adequate for intact sister chromatid alignment. In contrast to deletion of core cohesin ring components, which abrogates the survival of hematopoietic cells(19), somatic deletion of either Stag1 or Stag2 was compatible with hematopoietic stem cell viability, whereas loss of both genes led to hematopoietic failure and defective chromosomal segregation. Our in vivo studies underscore previous in vitro dependency studies(20) and suggest that therapies which directly or indirectly alter Stag1 function will induce a specific dependency in Stag2 mutant cancer cells. However, increased self-renewal, impaired lineage commitment, and progressive hematopoietic alterations were observed with Stag2 deletion but not with Stag1 loss. These studies illustrate that Stag2 has a key role in hematopoiesis and in hematopoietic transformation that is not shared by Stag1. Future studies will illustrate how Stag2 loss of function cooperates with other alleles to promote leukemic transformation and how co-occurring disease alleles cooperate with the chromatin architecture alterations induced by Stag2 loss.
Our data suggest that the role of Stag2 in regulating hematopoiesis and in hematopoietic transformation is Stag1-independent. Key hematopoietic regulators which show differential expression, accessibility, and insulation in the absence of Stag2 are bound by Stag2 and not by Stag1. This includes several key genes essential for lineage commitment to lymphoid fate, including the PU.1 targets Ebf1 and Pax5. Previous studies demonstrated that PU.1-mediated regulation of Ebf1 is a key event in B-cell lineage commitment(50, 51), and PU.1 has been defined to be a pioneer factor with nucleosome remodeling capacity. Given these data and the known role of Ebf1 in B cell commitment/differentiation (22), we show that PU.1 restoration cannot rescue the impact of Stag2 loss on hematopoietic self-renewal and lineage commitment without the permissive chromatin context regulated by Stag2. By contrast, restoration of Ebf1 expression restores differentiation and abrogates the aberrant self-renewal of Stag2 deficient cells.
Our data illustrate a key role for Stag2 in the balance between self-renewal and differentiation, a critical feature of transformation across different tumor types. Moreover, the observation that reactivation of the Stag2-target Ebf1 can reverse these alterations suggests a key role for Stag2-regulated transcription factor networks in tumor suppression, which is reversible when key networks are restored in Stag2 mutant cells. We hypothesize that Stag2 loss-of-function induces lineage specific alterations in transcription factor expression and function in a spectrum of malignant contexts. Most importantly, our studies identify a key pathophysiologic role for mutations in the factors that govern gene regulatory architecture in malignant transformation. Our data point to the reversibility of transcriptional dysregulation which mediates aberrant self-renewal and differentiation in cancer cells and suggest that therapies that reverse these alterations may have therapeutic benefit in cohesin-mutant cancers.
Funding
This work was supported by a National Cancer Institute R35 CA197594-01A1 (R.L.L.), National Cancer Institute R01 CA216421 (R.L.L.), National Cancer Institute PS-OC U54 CA143869-05 (R.L.L. and J.D.), and Leukemia & Lymphoma Society Translational Research Foundation 6499-17 (R.L.L.). A.D.V. is supported by a National Cancer Institute career development grant K08 CA215317, the William Raveis Charitable Fund Fellowship of the Damon Runyon Cancer Research Foundation (DRG 117-15), and an EvansMDS Young Investigator grant from the Edward P. Evans Foundation. R.L.B. is supported by the Sohn Foundation Fellowship of the Damon Runyon Cancer Research Foundation (DRG 22-17). T.B. is supported by the William C. and Joyce C. O’Neil Charitable Trust, Memorial Sloan Kettering Single Cell Sequencing Initiative. J.D. is an investigator of the Howard Hughes Medical Institute.
Author Contributions
Conceptualization, A.D.V., R.L.B., R.L.L. Methodology, A.D.V., R.L.B., Y.L., V.L., W.X., B.H.D., S.E.E., A.N., M.W., T.B., E.A., S.L., C.J.O., D.P., J.D., R.K., R.L.L. Investigation, A.D.V., R.L.B., Y.L., W.X., B.H.D., S.E.E., A.N., J.P., S.B., M.W., T.B., S.L., C.J.O., J.D., R.K., R.L.L. Writing – Original Draft, A.D.V., R.L.B. Writing – Review & Editing, A.D.V., R.L.B., Y.L., B.H.D., S.E.E., A.N., M.W., T.B., S.L., C.J.O., J.D., R.K., R.L.L. Funding Acquisition, R.L.L. Resources, J.D. R.L.L. Supervision, R.L.L.
Competing interests
A.D.V. received travel support from Mission Bio and is on the Editorial Advisory Board of Hematology News. J.D. is on the Scientific Advisory Board of Arima Genomics, and a consultant for Omega Therapeutics. R.L.L. is on the supervisory board of Qiagen and is a scientific advisor to Loxo, Imago, C4 Therapeutics and Isoplexis. He receives research support from and consulted for Celgene and Roche and has consulted for Janssen, Astellas, Morphosys and Novartis. He has received honoraria from Roche, Lilly and Amgen for invited lectures and from Gilead for grant reviews. R.L.B, Y.L., B.H.D., S.E.E., A.N., M.W., T.B., C.J.O., and R.K. have nothing to disclose.
Acknowledgments
We acknowledge the use of the Integrated Genomics Operation Core, funded by the Memorial Sloan Kettering Cancer Center Support Grant NIH P30 CA008748, and the Marie-Josée and Henry R. Kravis Center for Molecular Oncology. We would like to thank Dr. Gauri Nandakumar and the staff of the Cytogenetics Core Facility for their assistance in imaging and processing of cytogenetic data.