HMGA1 chromatin regulators induce transcriptional networks involved in GATA2 and proliferation during MPN progression

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell (HSC) disorders that are characterized by hyperactive JAK/STAT signaling and an increased risk for transformation to myelofibrosis (MF) and acute myeloid leukemia (AML).^1-18 However, actionable mechanisms driving progression remain elusive, and therapies are ineffective after leukemic transformation. The most common driver mutation, JAK2^V617F, enhances phosphorylation of STAT3/5 proteins, leading to overproduction of myeloid lineages.^19-22 Most patients present with chronic indolent disease and elevations in platelets (essential thrombocythemia; ET) or red cells and platelets (polycythemia vera; PV), although a subset presents with primary MF that is characterized by bone marrow (BM) and splenic fibrosis, osteosclerosis, splenomegaly, pancytopenia, and shortened life spans.^1-22 Although transformation to MF or AML is associated with acquisition of new mutations, therapies to prevent or treat transformation are lacking.^2-26 

Chromatin regulators maintain gene expression, conferring plasticity and other stem cell properties during development and oncogenic transformation.²⁷ The HMGA1 chromatin regulator gene is highly expressed in embryonic and adult stem cells, with low or undetectable levels in most differentiated tissues.^28-33 In diverse solid tumors, HMGA1 is aberrantly re-expressed; high levels are associated with adverse outcomes.^28-30,33-44 Located on chromosome 6p21, HMGA1 encodes HMGA1a/HMGA1b protein isoforms that bind to AT-rich regions in DNA, displacing repressive histones that compact chromatin.^{28,29,34,45-52} After binding DNA and “opening” the minor groove, HMGA1 recruits transcriptional complexes to modulate gene expression.^{28,29,34,45-52} Transgenic mice overexpressing Hmga1 in lymphoid cells develop clonal expansion and leukemia.^53-56,HMGA1 is also overexpressed in human lymphoid and myeloid malignancies.^53-61 Intriguingly, a recent study found that germline lesions within the HMGA1 loci predispose individuals to MPN.⁶² However, the role of HMGA1 in MPN had not been elucidated.

Here, we report that HMGA1 is upregulated and activates specific transcriptional networks during MPN progression. HMGA1 deficiency enhances sensitivity to the JAK inhibitor ruxolitinib and delays leukemogenesis in murine models of JAK2^V617 AML. Further, deletion of just a single Hmga1 allele prevents MF in JAK2^V617F mice. Our findings demonstrate that HMGA1 is a key epigenetic switch in MPN, thus opening the door to novel therapeutic approaches to treat or prevent progression.

Hmga1-deficient mice^44,63 and JAK2^V617F transgenic mice^64,65 were crossed to obtain the desired genotypes. We generated leukemia xenografts (DAMI/SET-2 cells; 1-5 × 10⁶) by tail vein injection into NOD Scid γ (NSG) mice. For transplantation studies, BM cells (4 × 10⁶) from 16-week donors (JAK2^V617F/V617F vs JAK2^V617F/V617F with Hmga1 heterozygosity [Hmga1^+/−]) were injected into lethally irradiated recipients (8 weeks; Ly5.1, 10 Gy).

Patient samples, protein and RNA analyses, chromatin immunoprecipitation sequencing (ChIPseq), chromatin immunoprecipitation polymerase chain reaction (ChIP-PCR), assay for transposase-accessible chromatin-sequencing (ATACseq), flow cytometry, immunohistochemistry, and cell-based assays were performed as described.^{31-33,40-44,53-61,66-72} RNA sequencing (RNAseq)/ATACseq and ChIP-PCR in parental DAMI cells (with and without HMGA1 silencing) were performed in triplicate. DAMI (American Type Culture Collection),^73,74 SET-2 (Leibniz Institute DSMZ),⁷⁵ and UKE-1 (Coriell Institute)⁷⁶ were cultured as recommended.

RNAseq of JAK2^V617F AML cells (DAMI/SET-2) was performed after transduction with control lentivirus or short hairpin RNA (shRNA) targeting HMGA1. Following data analyses, pathways were identified via MSig gene set enrichment analysis (GSEA).^77,78 Data from RNAseq, Gene Expression Omnibus data sets (GSE103237,⁷⁹ GSE122198,⁷¹ GSE144568⁷⁰), and peripheral blood mononuclear cells (PBMCs) from matched patients with MF that transformed to AML were analyzed as described.²⁶ Single-cell RNAseq (scRNAseq) was performed in BM-derived lineage⁻sca1⁺c-kit⁺ (LSK) cells from JAK2^V617F/V617F mice with both Hmga1 alleles intact or with Hmga1 heterozygous deficiency (2 mice per condition) and analyzed as described.^71,72

After ascertaining normal distributions (Ryan-Joyner, D’Agostino-Pearson tests), data were analyzed with a 2-tailed Student t test for 2 groups or 1-way analysis of variance (ANOVA), followed by Tukey’s multiple-comparison test; if not normal, we used the Mann-Whitney U test for 2 groups and the Kruskal-Wallis test, followed by Dunn’s test, for multiple comparisons. Cytotoxicity curves were analyzed by 2-way ANOVA. We compared survival using the Kaplan-Meier test, with the log-rank (Mantel-Cox) test and the Bonferroni method for multiple comparisons. Fisher’s exact test was used to compare differences in scRNAseq clusters across genotype. P < .05 was considered significant (GraphPad Prism 8.)

Additional details about methods and RNAseq analyses are provided in supplemental Methods (available on the Blood Web site).

To explore HMGA1 function in MPN, we first assessed gene expression in CD34⁺ cells from patients with MPN with acquired JAK2^V617F mutations (Figure 1A). Compared with unaffected individuals, HMGA1 was upregulated in peripheral blood (PB) CD34⁺ cells, with the highest levels after leukemic transformation. In a separate cohort (GSE103237),^79,HMGA1 was overexpressed in BM CD34⁺ cells from JAK2^V617F ET and PV. Intriguingly, HMGA1 was not overexpressed in CALR-mutant ET. By scRNAseq of PB CD34⁺ cells (GSE122198),^71,HMGA1 was also overexpressed in JAK2^V617F mutant cells from MF compared with unmutated cells from the same patients or unaffected individuals. HMGA1 overexpression in CD34⁺ cells from patients with MF compared with age-matched healthy individuals was confirmed by scRNAseq in another cohort (GSE144568⁷⁰). Together, these data demonstrate that HMGA1 is upregulated in JAK2^V617 MPNs.

Next, we disrupted gene expression via shRNA targeting 2 HMGA1 sequences or by CRISPR/Cas9 in JAK2^V617F AML cell lines (DAMI, SET-2, UKE-1).^73-76 Strikingly, depleting HMGA1 in all cell lines disrupts proliferation and clonogenicity, decreasing colony number and size (Figure 1B-D; supplemental Figure 1A-F). HMGA1 depletion also decreases the percentage of cells in S phase while increasing apoptosis (Figure 1E-F). Notably, HMGA2, a related HMGA family member, did not change with HMGA1 silencing (supplemental Figure 1A). Next, we compared leukemic engraftment following tail vein injection of AML cells into NSG mice. HMGA1 depletion in DAMI or SET-2 cells decreases spleen size, circulating blasts, and leukemic engraftment in BM and spleen 23 days following injection (Figure 1G-H; supplemental Figure 2A). BM architecture was generally normal in recipients of HMGA1-depleted cells, whereas architecture was effaced by leukemic blasts in recipients of AML cells lacking HMGA1 depletion. Survival was also prolonged in recipients of AML cells with HMGA1 silencing (Figure 1I; supplemental Figure 2A). Intriguingly, the small percentage of engrafted blasts from the pool of AML cells with HMGA1 silencing express higher HMGA1 than do the injected cells, suggesting that escape from gene silencing allows these cells to engraft (supplemental Figure 2B-C). Our findings indicate that HMGA1 is required for salient AML phenotypes, including proliferation, clonogenicity, and leukemic engraftment.

To investigate HMGA1 in models of chronic MPN, we crossed a well-characterized JAK2^V617F transgenic model^64,65 onto a background with global heterozygous Hmga1 deficiency.^44,63JAK2^V617F mice express 13 copies of the transgene and recapitulate features of chronic MPN with erythrocytosis and thrombocytosis by 12 weeks and progression to MF by 40 weeks, with osteosclerosis, splenomegaly, and fibrosis involving the spleen and BM (Figure 2; supplemental Figure 3A). Importantly, Hmga1 heterozygous mice have normal life spans and unperturbed hematopoiesis, based on steady-state blood counts, for up to 60 weeks (supplemental Figure 3B). Strikingly, loss of a single Hmga1 allele in JAK2^V617F mice prevents progression to MF, decreasing erythrocytosis by 12 weeks, and thrombocytosis, megakaryocyte hyperplasia, splenomegaly, fibrosis (spleen, BM) and osteosclerosis after 40 weeks (Figure 2).

To determine whether these effects depend on Hmga1 within the hematopoietic compartment, we transplanted BM from mice biallelic for the JAK2^V617F transgene (26 copies; denoted JAK2^V617F/V617F) with Hmga1 heterozygosity or both alleles intact into lethally irradiated recipients. As before, Hmga1 deficiency decreased erythrocytosis, thrombocytosis, megakaryocyte hyperplasia, splenomegaly, and fibrosis (spleen, BM) (Figure 3A-D), demonstrating that Hmga1 haploinsufficiency within hematopoietic cells is responsible for abrogating MF.

To determine how Hmga1 prevents MF, we assessed the frequency and function of hematopoietic stem cells (HSCs) and hematopoietic stem and progenitor cells (HSPCs). Long-term HSCs, LSK cells, and myeloid progenitors, including multipotent progenitors, granulocyte-monocyte progenitors (GMPs), and megakaryocyte-erythrocyte progenitors (MEPs) all decrease in BM from JAK^V617F/V617F transplants with Hmga1 haploinsufficiency. LSK cells, multipotent progenitors, common myeloid progenitors, GMPs, and MEPs decrease in spleen (Figure 3E), although there was no difference in common lymphoid progenitors (CLPs) in marrow or spleen (supplemental Figure 4). Hmga1 heterozygosity also dampens the clonogenic potential of JAK2^V617F/V617F BM and spleen cells, decreasing total colony-forming units, granulocyte-monocyte colony-forming units, and burst-forming unit-erythroid (supplemental Figure 5). These data demonstrate that Hmga1 haploinsufficiency within JAK^V617F/V617F HSCs dampens clonogenicity and expansion in HSCs and distinct progenitors.

To further characterize HSPCs regulated by Hmga1, we compared scRNAseq in BM-derived LSK cells from JAK2^V617F/V617F mice and JAK2^{V617F/JAKV617F} mice with Hmga1 heterozygous deficiency. Cell identify was assigned to clusters using published results.^72,80-83  Similar to our flow cytometry results, the cluster(s) enriched for markers of HSCs (cluster 7), megakaryocyte-biased progenitors (cluster 8), and megakaryocyte/erythroid-biased progenitors (clusters 5-6) all decreased in JAK2^V617F mice with Hmga1 heterozygous deficiency, whereas those with lymphoid-biased progenitor markers (clusters 0-1) increased (Figure 3F). These findings parallel our flow cytometry results, demonstrating that Hmga1 deficiency mitigates expansion in HSCs and distinct progenitors involved in MPN.

To further elucidate the molecular underpinnings of HMGA1, we performed RNAseq in DAMI and SET-2 cells and ChIPseq/ATACseq in DAMI cells. GSEA from RNAseq revealed an HMGA1 signature of genes involved in cell cycle progression (E2F targets, G2M checkpoint, mitotic spindle, MYC targets) and cell fate decisions (GATA2 transcriptional networks; Figure 4A-C). Ingenuity Pathway Analysis showed similar networks, in addition to those governing cell-cell signaling, inflammation, and fibrosis. To determine which genes could be direct targets of HMGA1, we integrated RNAseq with ChIPseq to detect differentially regulated genes with HMGA1 occupancy in potential gene-regulatory regions, which identified E2F targets, mitotic spindle, G2M checkpoint, and GATA2 networks (supplemental Table 1). ATACseq showed differentially enhanced chromatin accessibility in DAMI control cells compared with cells with HMGA1 depletion in promoters for genes regulating cell growth and signal transduction. Integrating RNAseq/ATACseq (by Enrichr; supplemental Table 2) revealed enrichment for GATA genes, KRAS signaling, and active histone marks H3 lysine 4 monomethylation (H3K4me1) or H3 lysine 4 trimethylation (H3K4me3). We focused on GATA2, given its robust regulation by HMGA1 in JAK2^V617F AML cells, role in HSC fate, and association with poor outcomes in myeloid malignancies.^84-87 HMGA1-dependent expression of GATA2 was validated (messenger RNA, protein) in DAMI, SET-2, and UKE-1 cells with HMGA1 deficiency via HMGA1 shRNA or CRISPR/Cas9 (Figure 4D-E; supplemental Figure 6A-B). Conversely, HMGA1 overexpression via CRISPR activation (HMGA1-CRISPRa) upregulates GATA2 (supplemental Figure 6C).

Next, we determined whether HMGA1 and GATA2 are coexpressed in wild-type (WT) or JAK2^V617F-mutant HSPCs. In unaffected human and murine HSPCs, HMGA1/Hmga1 and GATA2/Gata2 are positively correlated (supplemental Figure 6D-G). In JAK2^V617F murine HSPCs, Hmga1 and Gata2 are coexpressed and positively correlated, but only in LSK cells and MEPs (no correlation in common myeloid progenitors or GMPs; supplemental Figure 6H-I). Notably, LSK cell and MEP populations constitute MPN progenitors in JAK2^V617F models.⁸⁸ These findings demonstrate coexpression of HMGA1 and GATA2 in HSPCs and MPN progenitors in JAK2^V617F mice.

To determine whether HMGA1 directly activates GATA2, we identified 42 putative DNA binding sites throughout GATA2 regulatory regions using in silico algorithms, including 6 regions with ≥2 sites within 500 bp. HMGA1 chromatin occupancy at these 6 regions by ChIP-PCR and ChIPseq show the greatest enrichment around an established +9.5 GATA2 enhancer (Figure 4F-H) that is critical for HSC and MEP development during embryonic hematopoiesis.^84-86 Further, HMGA1 occupancy (ChIP-PCR) was depleted by HMGA1 silencing (Figure 4F-I). This +9.5 enhancer partially overlaps with a 224-bp sequence with 4 predicted HMGA1 binding sites (A, B, C, D). ChIP-PCR, used to compare occupancy around these sites, showed the greatest enrichment at sites A and B; recombinant HMGA1 binding by gel shift analysis was also most robust with probes within site A or B (Figure 4H-J; supplemental Figure 7A-E). Because HMGA1 induces GATA2 and associates with regions with active histone marks by ATACseq (H3K4me1, H3K4me3), we compared these marks by ChIP-PCR, which revealed enrichment at the +9.5 enhancer; both decreased upon HMGA1 depletion (Figure 4H-I). Conversely, there was no change in H3K27Ac or control histone H3 binding, suggesting that HMGA1 recruits H3K4me1/H3K4me3 in JAK2^V617F AML DAMI cells (supplemental Figure 7F-G).

To determine whether HMGA1 transactivates the GATA2 developmental enhancer, we assessed luciferase reporter gene activation of the +9.5 enhancer region upstream of the GATA2 minimal promoter (GATA2-1S). GATA2-1S showed low activity, which remained unchanged by HMGA1 silencing, whereas reporter activity increased more than three-fold when the +9.5 enhancer site A was present and more than four-fold when sites A and B were included; accordingly, +9.5 reporter activity decreased upon HMGA1 silencing (Figure 4K). Together, these results indicate that HMGA1 binds directly to sites near the GATA2 +9.5 enhancer, increasing chromatin accessibility and recruiting active histone marks (H3K4me1/H3K4me3) to induce GATA2.

To determine whether GATA2 is required for HMGA1 function, we depleted GATA2 in JAK2^V617F AML cell lines (DAMI, SET-2, and UKE-1) via shRNA targeting of 2 different sequences. Silencing GATA2 decreases proliferation, clonogenicity, and the frequency of cells in S phase while increasing apoptosis, recapitulating phenotypes observed with HMGA1 depletion (Figure 5A-F; supplemental Figure 8). GATA2 deficiency also disrupts the leukemogenic potential of DAMI cells, decreasing splenomegaly and leukemic engraftment (BM, spleen); moreover, survival was prolonged in recipients of DAMI cells with GATA2 silencing (Figure 5G-I). Similar to our results with HMGA1, engrafted leukemic cells from the pool of cells with GATA2 silencing express higher GATA2 compared with the injected cells, suggesting that a critical level of GATA2 is required for engraftment (supplemental Figure 9). Collectively, our results indicate that GATA2 is an important mediator of HMGA1 in these JAK2^V617F AML models.

To determine whether restoring GATA2 rescues anti-leukemogenic phenotypes mediated by HMGA1 silencing, we engineered DAMI, SET-2, and UKE-1 cells with HMGA1 silencing and concurrent expression of GATA2, such that GATA2 levels (messenger RNA, protein) approximate those of parental cells. GATA2 partially rescues phenotypes associated with HMGA1 depletion, increasing, but not restoring, clonogenicity in JAK2^V617F AML cell lines (Figure 6A-C; supplemental Figure 10A). GATA2 rescues the frequency of apoptotic cells to baseline levels, although proliferation and cell cycle progression were unchanged (Figure 6D; supplemental 10B-C). GATA2 restoration also partially rescues the antileukemogenic effects of HMGA1 silencing (Figure 6E-F). As before, disease latency was shortest in recipients of DAMI cells with endogenous expression of HMGA1 and GATA2 (median survival, 24 days) and was longest in recipients of cells with HMGA1 depletion (median survival, 38 days). The recipients of leukemic cells with HMGA1 silencing and GATA2 overexpression had intermediate survival (32.5 days; P < .01). GATA2 restoration increases the frequency of BM-engrafted DAMI cells more than two-fold compared with those with HMGA1 depletion, although splenic engraftment was unchanged (Figure 6F; supplemental Figure 10D). These results demonstrate that GATA2 partially mediates HMGA1 function in models of JAK2^V617F AML.

To determine the significance of the HMGA1-GATA2 network in human MPN progression, we compared the expression of HMGA1 and GATA2 in primary human MPN. Strikingly, GATA2 and HMGA1 are coexpressed and positively correlated via RNAseq from PBMCs from a cohort of matched patients with MF that transformed to AML (n = 11) (Figure 6Gi-ii).²⁶ Further, this relationship was validated in independent studies (Figure 6Giii-iv; supplemental Figure 11): (1) HMGA1 and GATA2 are coexpressed and upregulated in BM CD34⁺ cells with progression from PV or ET to MF (GSE103237),⁷⁹ (2) HMGA1 and GATA2 are upregulated in PB CD34⁺ cells from MF compared with age-matched healthy controls by scRNAseq (GSE144568),⁷¹ and, (3) in another scRNAseq cohort with greater sequencing depth, HMGA1 and GATA2 are coexpressed and upregulated in JAK2-mutated PB CD34⁺ cells from MF compared with unmutated CD34⁺ cells from the same patients or CD34⁺ cells from unaffected individuals (GSE122198).⁷⁰ 

Next, we identified the pathways that are activated in human MPN. In the matched cohort of MF to AML, most leukemic samples show activation of pathways distinct from those in MF, and there was a striking overlap with pathways activated in leukemia and HMGA1 networks identified in our cell-based models. Specifically, networks driving cell cycle progression pathways are activated after transformation to AML, including the most significant HMGA1 pathways (G2M, mitotic spindle, E2F; Figure 6H). Pathways repressed in the leukemic samples overlap with those repressed by HMGA in JAK2^V617F AML cell-based models, including interferon-γ (data not shown). Furthermore, HMGA1 transcript levels correlate with HMGA1 pathway activation in the primary MPN AML cells (Figure 6H). Similarly, the scRNAseq cohorts show activation of HMGA1 networks (E2F targets, G2M checkpoint, DNA repair), including GATA2 with progression to MF.^70,71 Together, these results implicate HMGA1 as a driver of transcriptional networks required in human MPN progression.

Next, we determined whether HMGA1 deficiency enhances sensitivity to the JAK/STAT inhibitor ruxolitinib in JAK2^V617F AML. First, we found that the concentration at which 50% of DAMI cells stop proliferating with ruxolitinib treatment decreases upon HMGA1 depletion (406.5 nM vs 204.8 nM; P < .001) (Figure 7A). Second, we tested the effects of ruxolitinib with HMGA1 silencing on leukemogenesis in vivo. Therapy was delivered via chow supplemented with ruxolitinib (2000 mg INCB01842 per kilogram diet) to provide doses shown to reach therapeutic levels in mice^89,90; control chow was identical except it lacked ruxolitinib. We included 4 groups: (1) DAMI control, control chow, (2) DAMI control, ruxolitinib chow, (3) DAMI with HMGA1 depletion, control chow, and (4) DAMI with HMGA1 depletion, ruxolitinib chow. After 24 days of therapy, we assessed leukemic engraftment. Mice injected with control DAMI cells and placed on control chow had the highest leukemic burdens in spleen and BM, whereas recipients of DAMI cells with HMGA1 silencing and ruxolitinib treatment showed the lowest engraftment and spleen sizes, followed by recipients of DAMI cells with HMGA1 silencing and control chow (Figure 7B-C; supplemental Figure 12). Moreover, survival is prolonged in recipients of AML cells with HMGA1 silencing and ruxolitinib therapy (Figure 7D).

We also tested ruxolitinib in the JAK^V617F/V617F chronic MPN model with therapy beginning at 16 to 20 weeks of age when erythrocytosis and splenomegaly are well established (supplemental Figure 13A). Ruxolitinib decreased splenomegaly and fibrosis in JAK^V617F/V617F mice (Figure 7E-F). In the JAK^V617F/V617F model, Hmga1 heterozygosity decreases splenomegaly and prevents fibrosis (supplemental Figure 13B). Spleen sizes decrease further with ruxolitinib in JAK^V617F/V617F mice with Hmga1 heterozygosity (supplemental Figure 13). There were decreases in leukocytosis with modest changes in erythrocytosis in both models (supplemental Figure 13A). Together, these results indicate that HMGA1 silencing or Hmga1 haploinsufficiency enhances sensitivity to ruxolitinib in preclinical MPN models and strongly support HMGA1 as a rational therapeutic target.

We discovered a novel role for HMGA1 as an epigenetic switch involved in JAK2^V617F MPN progression. Using shRNA-mediated or CRISPR/Cas9-mediated gene silencing in JAK2^V617F models, we found that HMGA1 deficiency: 1) decreases proliferation, clonogenicity, and leukemic engraftment; 2) represses transcriptional networks involved in cell cycle progression, proliferation, and GATA2 function; and, 3) most importantly, enhances sensitivity to the JAK inhibitor ruxolitinib in disrupting leukemic engraftment and prolonging survival. We validated the significance of HMGA1 in human MPN because HMGA1 and downstream pathways, including GATA2, are upregulated during progression in independent cohorts. Moreover, HMGA1 transcriptional pathways are activated in JAK2^V617F CD34⁺ cells compared with those lacking mutant JAK, and in matched MF samples after transformation to leukemia, further illuminating HMGA1 as a promising therapeutic target. The enhanced response to ruxolitinib by HMGA1 depletion is particularly striking in AML because ruxolitinib is not effective in patients with MPN after leukemic transformation.

Surprisingly, Hmga1 haploinsufficiency prevents MF in chronic MPN models, reducing erythrocytosis, megakaryocyte hyperplasia, splenomegaly, and fibrosis, while dampening expansion of JAK2^V617F mutant stem and progenitor cells. This was unexpected for 2 reasons; first, although HMGA1 is a potent oncogene and driver of progression in aggressive solid tumor modes, its role in clonal hematopoiesis and more indolent neoplasia had not been demonstrated. Second, most studies focusing on HMGA1 in tumor cells rely on gene silencing to low levels. Thus, we did not anticipate that reducing levels by only 50% would so profoundly impact progression. Moreover, the normal steady-state hematopoiesis in Hmga1 heterozygous mice suggests that pharmacologic modulation of HMGA1 to this degree is a plausible therapeutic end point, although specific inhibitors of HMGA1 are not yet available.

To uncover actionable mechanisms mediated by HMGA1, we investigated downstream transcriptomic and epigenetic changes, which identified HMGA1-dependent transcriptional networks governing cell cycle progression and cell fate, including E2F and GATA2 targets. Intriguingly, a recent study using mutant MPL^W515L murine models identified E2F networks in MPN progression that were targeted by inhibiting PRMT5, a protein arginine methyltransferase,²⁵ although it remains to be shown whether similar approaches disrupt HMGA1 networks. Instead, we focused on the GATA2 master regulator, given its association with HSC function and poor outcomes in AML.^84-87 

HMGA1 aberrantly induces GATA2 in human JAK^V617F AML cell lines and murine models. Moreover, this pathway is activated in human MPN progression. HMGA1-mediated induction of GATA2 could preclude normal GATA1 function in HSPCs and megakaryocytes, thereby mimicking the GATA1-low phenotype that causes MF in mice.^91-93 Low GATA1 protein levels were observed in megakaryocytes from patients with MF.^92,93 We did not test the effects of Gata2 loss in our JAK^V617F mice because Gata2 deficiency disrupts hematopoiesis. Modulating HMGA1 provides a more attractive therapeutic approach. Notably, repressing GATA2 recapitulates many phenotypes observed with HMGA1 silencing, and restoring GATA2 only partially rescues anti-leukemic effects of HMGA1 depletion, demonstrating that GATA2 is only one HMGA1 effector in MPN. Indeed, RNAseq, ChIPseq and ATACseq revealed other networks that are involved in proliferation; thus, pharmacologically targeting these pathways may be beneficial.

The role of HMGA1 in orchestrating the assembly of specific histone marks had not been studied in detail. We found that HMGA1 enhances chromatin accessibility and recruits histone marks associated with gene activation, including H3K3me3 (for active promoters) and H3K4me1 (active enhancers). Previous studies focused on HMGA1 occupancy upstream of minimal promoters,^28,29 although we discovered HMGA1 occupancy at the GATA2 +9.5 distal enhancer, suggesting that HMGA1 activates or “rewires” enhancer function when overexpressed during MPN progression.

In JAK2^V617F mice, Hmga1 haploinsufficiency mitigates expansion of HSCs, specific myeloid progenitors (MEPs, GMPs), and megakaryocytes, without affecting CLPs. scRNAseq corroborated these results, indicating that Hmga1 haploinsufficiency decreased clusters with HS and megakaryocyte/erythroid-biased markers while expanding those with lymphoid-biased markers. HSCs and MEPs represent key progenitors in diverse MPN models of MF, including mice expressing JAK2^V617F or mutant thrombopoietin receptor MPL^W515L.^23,94 HMGA1 is also a master regulator in other adult stem cell populations, including intestinal stem cells,^28,33 which rely on many transcription factors that are active in HSCs. Further, intestinal epithelium and hematopoiesis require highly regenerative stem cell pools to replace mature progeny, and HMGA1 is re-expressed in hematologic and intestinal malignancies where it amplifies stem-like networks.^28,33,54 These data suggest that tightly regulated HMGA1 is critical for stem cell function under homeostatic conditions, whereas mutated genomes, together with HMGA1 overexpression, foster aberrant proliferation, differentiation, and transformation.

Intriguingly, HMGA1 was upregulated in early-stage MPN with JAK2 mutations, but not in CALR-mutated early-stage disease. In our Johns Hopkins University cohort, CD34⁺ AML blasts with the highest HMGA1 expression also harbor mutant EZH2 and ASXL1, suggesting that other clonal hematopoiesis lesions may induce HMGA1. Given the prevalence of clonal hematopoiesis with aging, further studies to investigate HMGA1 in this setting are warranted. Although HMGA2 has been studied in myeloid malignancy and clonal hematopoiesis,^95-98 RNAseq revealed barely detectable HMGA2 transcripts in human DAMI and SET-2 cell lines and very low levels in most primary human MPN samples. Moreover, Hmga2 deficiency did not impact progression in the JAK2^V617F model when crossed to mice with homozygous or heterozygous deficiency of Hmga2. Together, our studies implicate HMGA1, rather than HMGA2, as a primary driver of JAK2^V617F MPN progression.

In summary, we report a novel role for HMGA1 in JAK2^V617F AML and MPN progression using diverse models and orthogonal approaches. Mechanistically, HMGA1 induces transcriptional networks that are involved in cell cycle progression and aberrant differentiation, the latter of which includes direct transactivation of GATA2. Together, our findings reveal HMGA1 as a new therapeutic target in MPN and open the door to novel therapeutic approaches to prevent disease progression. Because HMGA1 and GATA2 are also overexpressed in de novo AML, this pathway may be relevant, more broadly, in myeloid malignancies.

The authors thank the Mutation Generation and Detection Core at the University of Utah (National Institute of Diabetes and Digestive and Kidney Diseases Cooperative Hematology Specialized Core Center Grant U54DK110858), the Yale Cooperative Centers of Excellence in Hematology (National Institute of Diabetes and Digestive and Kidney Diseases Grant U54DK106857), and the Johns Hopkins University Ross Flow Core (National Institutes of Health Shared Instrument Grants S10OD026859 for Cytek Aurora Full Spectrum Flow Cytometric Analyzer).

This work was supported by National Institutes of Health National Heart, Lung, and Blood Institute grants R01HL145780 (L.M.S.R., A.R.M.) and R01HL143818 (L.M.S.R.), National Institutes of Health National Cancer Institute grant CA232741 (L.M.S.R.), National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases grant DK102943 (L.M.S.R.), the MPN Research Foundation (L.R., A.R.M.), the Children’s Cancer Research Fund (L.M.S.R.), Alex’s Lemonade Stand Foundation (L.M.S.R.), the Allegheny Health Network (L.M.S.R.), and the Maryland Stem Cell Research Fund (2021-MSCRFD-5697 and 2020-MSCRFD-5372) (L.M.S.R.).

Contribution: L.L. and J.-H.K. designed experiments, performed research, interpreted data, and wrote portions of the manuscript; W.L., D.M.W., and J.K. performed experiments and interpreted data; L.C. performed biostatistical analyses and wrote portions of the manuscript; R.K.R. and R.P.K. provided critical reagents and data; L.X. and L.Z.L. performed experiments; M.V. performed Ingenuity Pathway Analysis analyses; D.R.M. provided the GATA2 antibody and helped to edit the manuscript; Z.J.Z. provided JAK-mutant mice; O.R. performed experiments; M.C.S. provided ruxolitinib chow; K.R. provided guidance with ATACseq and chromatin immunoprecipitation experiments; A.-R.R. and B.P. performed scRNAseq analysis of patient samples; J.L.S. provided expertise and reagents and guidance for experiments and helped to write the manuscript; A.R.M. provided expertise and reagents, designed and performed experiments, and helped to write the manuscript; L.M.S.R. conceived the study, designed experiments, reviewed all data, and wrote the manuscript; and all authors reviewed the final manuscript and agreed to be coauthors.

Conflict-of-interest disclosure: M.C.S. is employed by Incyte Research Institute and holds individual shares in this privately held company. R.K.R has received consulting fees from Stemline Therapeutics, Celgene Corporation, Agios Pharmaceuticals, Apex Oncology, Beyond Spring, Partner Therapeutics, and Jazz Pharmaceuticals and has received research funding from Constellation Pharmaceuticals, Incyte Corporation, and Stemline Therapeutics. The remaining authors declare no competing financial interests.

Correspondence: Linda M. S. Resar, The Johns Hopkins University School of Medicine, Ross Research Building, Room 1025, 720 Rutland Ave, Baltimore, MD 21205; e-mail: lresar@jhmi.edu.