Abstract Background: Uterine serous carcinoma (USC) is an aggressive uterine cancer subtype that is responsible for 40% of uterine cancer deaths. We recently demonstrated that USCs lose expression of the zinc-finger transcription factor GATA2 which drives USC invasion, predicts USC recurrence, and is closely correlated with poor cancer-related and overall survival. However, the mechanistic basis of GATA2 downregulation in USC remains unknown. Methods: GATA2 RNA transcripts were correlated to patient outcomes derived from The Cancer Genome Atlas (TCGA). GATA2 gene methylation was assessed in isolated genomic DNA from patient USC tumors by bisulfite sequencing. Ark1 and Ark2 USC cell lines were utilized for in vitro studies. GATA2 half-life was measured using cycloheximide chase experiments paired with inhibitors of phosphorylation (staurosporine), acetylation (C646), SUMOylation (ML792), cysteine peptidase activity (N-Ethylmaleimide/NEM), and proteasome-dependent degradation (MG132). Anti-GATA2 immunoprecipitations were performed with custom anti-GATA2 antibodies ( Im et al, 2005). Depletion of PIAS2, SENP1, and SUMO2/3 was performed using commercially available siRNAs. Levels of GAPDH, GATA2, PIAS2, SENP1, and SUMO2/3 were measured by western blotting. USC invasion was assessed using Matrigel-coated membrane transwell inserts. Results: GATA2 IHC protein levels showed no correlation with GATA2 gene body or proximal promoter DNA methylation, and GATA2 transcript levels did not predict patient outcome across TCGA USC cases. Post-translationally, USC GATA2 protein half-life was approximately 60 minutes after cycloheximide treatment. Co-treatment with the SUMOylation inhibitor ML792 or the proteasome inhibitor MG132 prolonged GATA2 half-life compared with vehicle, whereas NEM treatment, which inhibits de-SUMOylation and de-ubiquitination, shortened GATA2 half-life. Direct GATA2 SUMOylation was confirmed by anti-GATA2 immunoprecipitation and SUMO2/3 western blot. A candidate siRNA-based approach found that depletion of the E3 SUMO ligase PIAS2 elevated GATA2 levels, while depletion of the SUMO peptidase SENP1 reduced GATA2 levels. siRNA-mediated SUMO2/3 depletion significantly increased GATA2 levels in USC cells and suppressed USC invasion in vitro compared to siScramble controls. Conclusion: USC GATA2 levels are determined by post-translational mechanisms. GATA2 protein has a rapid 60-minute half-life determined by SUMOylation and proteasome-mediated degradation. Depletion experiments support PIAS2 as the GATA2-targeting E3 SUMO ligase and SENP1 as the GATA2-targeting SUMO peptidase. SUMO inhibition increased USC GATA2 levels and suppressed USC invasion, suggesting that SUMO-targeting agents may suppress USC spread through upregulation of GATA2 levels. Citation Format: Anuoluwapo A. Mattix, Peng Liu, Molly A. Accola, William M. Rehrauer, Daniel R. Matson. SUMOylation regulates GATA2 stability to control uterine serous carcinoma invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3467.

Uterine cancer is the 4th most common cancer in women. Its incidence is increasing and its mortality rate also is rising. Uterine serous carcinoma (USC) is an aggressive uterine cancer subtype that is responsible for 40% of uterine cancer deaths. Although 50% of USC patients present with cancer localized to the uterine body (FIGO stage I) and should be cured by staging hysterectomy, 30% of these patients later recur in the absence of adjuvant combination paclitaxel-carboplatin chemotherapy. There is no reliable method to identify which USC patients are at risk of USC recurrence, so most patients receive risk-reducing adjuvant chemotherapy associated with significant morbidity. A priori knowledge of recurrence risk would enable a risk-stratified approach to adjuvant therapy to reduce morbidity and optimize survival. GATA Binding Protein 2 (GATA2) is a transcription factor that supports progesterone receptor signaling in the uterus. We recently generated anti-GATA2 monoclonal antibodies that can routinely detect GATA2 by standard immunohistochemistry (IHC). We assembled a retrospective multi-institutional cohort of 81 patients with primary FIGO stage I USCs. Associations between GATA2 levels and clinicopathologic metrics were evaluated using Student’s t-test, Fisher’s exact test, Kaplan-Meier method, and Cox proportional hazards ratio. Gene targets including GATA2 were depleted from Ark1 and Ark2 patient-derived USC cell lines using siRNA or shRNA, and cell invasion evaluated using Matrigel-coated membranes and organoid-based natural hydrogel. RNA-seq, anti-GATA2 ChIP-seq, and western blotting enabled identification of GATA2 gene targets. GATA2 expression in USC tumors ranged from 0-100% GATA2+ tumor nuclei. USCs with >15% GATA2+ nuclei were defined as GATA2-high based on an initial inflection point in the data that optimally delineated patients by outcome. The 39% of patients with GATA2-high USCs had 100% recurrence-free, 100% disease-specific, and 87% overall survival. These outcomes were significantly better than patients with GATA2-low USCs. Depletion of GATA2 in USC cell lines increased invasion in vitro. In patients who received no adjuvant chemotherapy (n=18), those with GATA2-high USCs had 100% recurrence-free survival compared to 50% recurrence-free survival in GATA2-low USC patients. A multi-omic approach identified SIN3 Transcription Regulator Family Member B as a GATA2 target gene that suppressed USC invasion in vitro. GATA2 IHC identifies FIGO stage I USC patients with a greatly reduced risk of USC recurrence. A GATA2 guided personalized medicine approach using standard IHC methods could be rapidly implemented in most hospital settings, would reduce treatment-related morbidity, and may optimize survival for USC patients. Usha S. Polaki, Trey E. Gilpin, Apoorva T. Patil, Emily Chiu, Paula M. Manan Mejias, Pei Hui, Maria Virumbrales-Muñoz, Lisa Barroilhet, Stephanie M. McGregor, Emery H. Bresnick, Daniel R. Matson. Loss of GATA2 promotes invasion and predicts cancer recurrence and survival in FIGO stage I uterine serous carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3349.

BACKGROUNDA priori knowledge of recurrence risk in patients with nonmetastatic (International Federation of Gynecology and Obstetrics [FIGO] stage I) uterine serous carcinoma (USC) would enable a risk-stratified approach to the use of adjuvant chemotherapy. This would greatly reduce treatment-related morbidity and be predicted to improve survival.METHODSGATA2 expression was scored by IHC across a retrospective multiinstitutional cohort of 195 primary USCs. Associations between GATA2 levels and clinicopathologic metrics were evaluated using Student's t test, Fisher's exact test, Kaplan-Meier method, and Cox proportional hazard ratio. Invasion in patient-derived USC cells was assessed by Student's t test. RNA-Seq, anti-GATA2 ChIP-Seq, and confirmatory Western blotting enabled identification of GATA2 targets.RESULTSPatients with FIGO stage I GATA2hi USCs had 100% recurrence-free and 100% cancer-related survival, which was significantly better than patients with GATA2lo USCs. In patients for whom adjuvant chemotherapy was omitted, patients with GATA2hi USC had 100% recurrence-free 5-year survival compared with 60% recurrence-free survival in patients with GATA2lo USC. Depletion of GATA2 in patient-derived USC cells increased invasion in vitro.CONCLUSIONRoutine GATA2 IHC identifies 33% of patients with FIGO stage I USC who have a greatly reduced risk of posthysterectomy USC recurrence. Our results suggest that a GATA2-guided personalized medicine approach could be rapidly implemented in most hospital settings, would reduce treatment-related morbidity, and would likely improve outcomes in patients with USC.FUNDINGNIH grants R01 DK068634, P30 CA014520, S10 OD023526, K08 DK127244, T32 HL007899, the UW-Madison Department of Pathology and Laboratory Medicine, the UW-Madison Centennial Scholars Program, the Diane Lindstrom Foundation, the American Cancer Society, the V Foundation, The Hartwell Foundation, and the UMN Department of Obstetrics, Gynecology, and Women's Health.

Piezo1 is a mechanosensitive ion channel that couples extracellular matrix (ECM) properties to intracellular signaling cascades that can modulate cell phenotypes. Piezo1 was recently shown to regulate metastasis in high-grade ovarian serous carcinoma (HGSOC) by promoting tumor budding in an ECM stiffness-dependent manner. Uterine serous carcinoma (USC) is an aggressive gynecologic malignancy that is increasing in incidence and responsible for 40% of uterine cancer deaths. USC rapidly invades and metastasizes via mechanisms that remain incompletely understood. Whether Piezo1 may have a similar function in USC, which shares many molecular and clinical features of HGSOC, is not known. We recently showed that expression of the transcription factor GATA2 is lost in USCs that locally invade into the wall of the uterus versus non-invasive USCs. However, the transcriptional targets of GATA2 that mediate this activity are not known. We performed RNAseq in patient-derived USC cells lines after GATA2 depletion to identify differentially expressed genes. We performed anti-GATA2 ChIPseq in patient derived USC cells to identify genome wide sites of GATA2 occupancy. Western blots were performed for GATA2, Piezo1, and Tubulin after GATA2 depletion in patient-derived USC cells modified to express doxycycline-inducible anti-GATA2 shRNAs or shScramble control. In vitro models of ECM with different stiffnesses (3.7 and 14 kPa) were generated using an interpenetrating network of collagen I and methacrylated gelatin (GelMA), crosslinked within transwell inserts. USC cells were seeded on top of the gels and monitored over 24 hours for invasion in response to GATA2 depletion. GATA2 depletion led to an increase in Piezo1 transcripts in patient-derived USC cells (log2fold change = 0.28, FDR<0.005), while western blot revealed a significant increase in Piezo1 protein expression after GATA2 depletion in two unique patient-derived USC cell lines. Anti-GATA2 ChIPseq identified two significant peaks of GATA2 occupancy within intron 1 of the Piezo1 gene, each harboring conserved canonical GATA motifs. Depletion of GATA2 in patient-derived USC cells led to increased tumor cell invasion, but only on the stiffer substrates. GATA2 binds two putative GATA enhancers within intron 1 of the Piezo1 gene in USC. GATA2 depletion leads to increased Piezo1 RNA and protein expression, which is accompanied by increased tumor cell invasion in a stiffness-dependent manner. Our findings support a model where loss of GATA2-mediated Piezo1 suppression facilitates increased Piezo1 expression, and Piezo1-driven ECM stiffness-dependent USC tumor invasion. They justify further studies to evaluate whether a GATA2-Piezo1 axis is a critical driver of USC invasion in vivo. Brittany Baikie, Mayuri Dutta, Pamela K. Kreeger, Daniel R. Matson. GATA2 loss promotes Piezo1 expression and stiffness-dependent invasion in uterine serous carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2658.

ABSTRACT:GATA binding protein 2 (GATA2) is a conserved zinc finger transcription factor that regulates the emergence and maintenance of complex genetic programs driving development and function of hematopoietic stem and progenitor cells (HSPCs). Patients born with monoallelic GATA2 mutations develop myelodysplastic neoplasm (MDS) and acute myeloid leukemia (AML), whereas acquired GATA2 mutations are reported in 3% to 5% of sporadic AML cases. The mechanisms by which aberrant GATA2 activity promotes MDS and AML are incompletely understood. Efforts to understand GATA2 in basic biology and disease will be facilitated by the development of broadly efficacious antibodies recognizing physiologic levels of GATA2 in diverse tissue types and assays. Here, we purified a polyclonal anti-GATA2 antibody and generated multiple highly specific anti-GATA2 monoclonal antibodies, optimized them for immunohistochemistry on patient bone marrow bioosy samples, and analyzed GATA2 expression in adults with healthy bone marrow, MDS, and acute leukemia. In healthy bone marrow, GATA2 was detected in mast cells, subsets of CD34+ HSPCs, E-cadherin-positive erythroid progenitors, and megakaryocytes. In MDS, GATA2 expression correlates with bone marrow blast percentage, positively correlates with myeloid dysplasia and complex cytogenetics, and is a nonindependent negative predictor of overall survival. In acute leukemia, the percent of GATA2+ blasts closely associates with myeloid lineage, whereas a subset of lymphoblastic and undifferentiated leukemias with myeloid features also express GATA2. However, the percent of GATA2+ blasts in AML is highly variable. Elevated GATA2 expression in AML blasts correlates with peripheral neutropenia and complex AML cytogenetics but, unlike in MDS, does not predict survival.

The GATA gene family encodes highly conserved zinc-finger transcription factors that facilitate the development and function of multiple organ systems including the uterus. In the endometrium, GATA2 functions in a positive autoregulatory loop with the progesterone receptor (PGR) and colocalizes with PGR on chromatin to promote PGR transcriptional programs. GATA2 also has PGR-independent functions that maintain endometrial cell identity, and GATA2 transcripts reportedly are down-regulated in endometrial disorders including endometriosis. This event is accompanied by a reciprocal increase in GATA6. Here, we applied custom anti-GATA2 monoclonal antibodies and performed GATA2 immunohistochemistry (IHC) on patient endometrial tissues corresponding to proliferative, secretory, inactive, and hormone-treated endometrium, as well as endometriosis and endometrial atypical hyperplasia/endometrioid intraepithelial neoplasia (EAH/EIN). We also performed IHC for the estrogen receptor, PGR, and GATA6 in relevant groups. The results reveal a tight correlation between GATA2 and PGR expression in the glandular and stromal cells of benign endometrium. GATA2 expression is markedly reduced in stromal but not glandular cells in endometriosis and EAH/EIN. This reduction in GATA2 expression does not lead to a detectable increase in GATA6 expression in endometriosis. Although average glandular GATA2 expression was preserved in endometriosis and EAH/EIN cases, its expression was decoupled from PGR, implying that alternative pathways regulate GATA2 levels in these disorders. Our findings indicate that GATA2 dysregulation is a feature of endometriosis and EAH/EIN, and support a model whereby loss of stromal GATA2 in these disorders contributes to their progesterone insensitivity.

Dynein inactivates the spindle assembly checkpoint (SAC) by transporting check-point proteins away from kinetochores toward spindle poles in a process known as "strip-ping." We find that inhibition of Aurora A kinase, which is localized to spindle poles, enables the accumulation of the spindle checkpoint activator Mad1 at poles where it is normally ab-sent. Aurora kinases phosphorylate the dynein activator NudE neurodevelopment protein 1 like 1 (Ndel1) on Ser285 and Mad1 accumulates at poles when Ndel1 is replaced by a non-phosphorylatable mutant in human cells. The pole focusing protein NuMA, transported to poles by dynein, also accumulates at poles in cells harboring a mutant Ndel1. Phosphoryla-tion of Ndel1 on Ser285 is required for robust spindle checkpoint activity and regulates the poles of asters in Xenopus extracts. Our data suggest that dynein/SAC complexes that are generated at kinetochores and then transported directionally toward poles on microtubules are inhibited by Aurora A before they reach spindle poles. These data suggest that Aurora A generates a spatial signal at spindle poles that controls dynein transport and spindle function.

Pilomatricomas (PMs) are common benign adnexal tumors that show a predilection for the head and neck region and are characterized at the molecular level by activating mutations in the beta-catenin (CTNNB1) gene. Giant PMs are a rare histopathological variant, according to the World Health Organization, which are defined by a size greater than 4 cm and are reported to show upregulation of yes-associated protein compared to PMs of typical 1-3 cm size. We describe the case of a 67-year-old man with an 8 cm giant PM involving his temporal scalp, whose PM we characterized by 10X spatial gene expression analysis. This revealed five total transcriptomic clusters, including four distinct clusters within the giant PM, each with a unique transcriptional pattern of hair follicle-related factors, keratin gene expression, and beta-catenin pathway activity.

Myeloid malignancies associated with germline predisposition syndromes account for up to 10% of myeloid neoplasms. They are classified into three categories by the proposed 5th Edition of the World Health Organization Classification of Hematolymphoid Tumors: (1) neoplasms with germline predisposition without a pre-existing platelet disorder or organ dysfunction, (2) neoplasms with germline predisposition and pre-existing platelet disorder, or (3) neoplasms with germline predisposition and potential organ dysfunction. Recognizing these entities is critical because patients and affected family members benefit from interfacing with hematologists who specialize in these disorders and can facilitate tailored treatment strategies. However, identification of these syndromes in routine pathology practice is often challenging, as characteristic findings associated with these diagnoses at baseline are frequently absent, nonspecific, or impossible to evaluate in the setting of a myeloid malignancy. Here we review the formally classified germline predisposition syndromes associated with myeloid malignancies and summarize practical recommendations for pathologists evaluating a new myeloid malignancy diagnosis. Our intent is to empower clinicians to better screen for germline disorders in this common clinical setting. Recognizing when to suspect a germline predisposition syndrome, pursue additional ancillary testing, and ultimately recommend referral to a cancer predisposition clinic or hematology specialist, will ensure optimal patient care and expedite research to improve outcomes for these individuals.

Diffuse large B-cell lymphoma, not otherwise specified (DLBCL NOS) is the most common lymphoid malignancy in the Western world and classically presents as a rapidly enlarging nodal or extranodal mass. Cutaneous involvement by systemic DLBCL NOS is an infrequent clinical presentation, encountered in only 1.5-3.5% of cases, while disseminated cutaneous disease with multiple subcutaneous nodules at the time of diagnosis is unusual and can present a diagnostic challenge. The differential diagnosis when encountering a high-grade B-cell malignancy at a cutaneous site is broad and includes primary cutaneous follicle center lymphoma (PCFCL), primary cutaneous diffuse large B-cell lymphoma, leg type (PCDLBCL-LT), high-grade B-cell lymphoma with MYC and BCL2 rearrangements (HGBCL-MYC/BCL2), and other potential entities which must all be carefully considered before rendering a final diagnosis. In this report, we describe the case of a 69-year-old man who was seen at our hospital due to generalized weakness and was found to have multiple subcutaneous nodules representing disseminated DLBCL NOS. The case was complicated by concurrent monoclonal B-cell lymphocytosis involving the bone marrow.

We previously demonstrated that a subset of acute myeloid leukemia (AML) patients with concurrent RAS pathway and TP53 mutations have an extremely poor prognosis and that most of these TP53 mutations are missense mutations. Here, we report that, in contrast to the mixed AML and T cell malignancy that developed in NrasG12D/+ p53–/– (NP–/–) mice, NrasG12D/+ p53R172H/+ (NPmut) mice rapidly developed inflammation-associated AML. Under the inflammatory conditions, NPmut hematopoietic stem and progenitor cells (HSPCs) displayed imbalanced myelopoiesis and lymphopoiesis and mostly normal cell proliferation despite MEK/ERK hyperactivation. RNA-Seq analysis revealed that oncogenic NRAS signaling and mutant p53 synergized to establish an NPmut-AML transcriptome distinct from that of NP–/– cells. The NPmut-AML transcriptome showed GATA2 downregulation and elevated the expression of inflammatory genes, including those linked to NF-κB signaling. NF-κB was also upregulated in human NRAS TP53 AML. Exogenous expression of GATA2 in human NPmut KY821 AML cells downregulated inflammatory gene expression. Mouse and human NPmut AML cells were sensitive to MEK and NF-κB inhibition in vitro. The proteasome inhibitor bortezomib stabilized the NF-κB–inhibitory protein IκBα, reduced inflammatory gene expression, and potentiated the survival benefit of a MEK inhibitor in NPmut mice. Our study demonstrates that a p53 structural mutant synergized with oncogenic NRAS to promote AML through mechanisms distinct from p53 loss.

GATA binding factor 2 (GATA2) is a zinc finger transcription factor that regulates complex genetic programs to promote development of the hematopoietic system in the embryo and proper function of the bone marrow in adults. GATA2 is upregulated at the RNA transcript level in myelodysplastic syndrome (MDS), a heterogeneous group of clonal hematopoietic stem cell malignancies characterized by peripheral cytopenias, ineffective hematopoiesis, genetic mutations, and a predisposition to develop acute myeloid leukemia (AML). We sought to directly visualize GATA2 protein expression in benign and MDS bone marrow core biopsies from human patients to identify the cellular compartments that express GATA2 protein and to compare GATA2 protein expression in benign bone marrows to bone marrows involved by MDS. We optimized an affinity purified polyclonal rabbit anti-GATA2 antibody for immunohistochemistry (IHC) on decalcified human patient bone marrow core biopsies and established dual-IHC protocols for GATA2 and CD34, CD117/c-Kit (CD117), E-Cadherin (Ecad), and Myeloperoxidase (MPO). GATA2 expression was then scored and compared to clinicopathologic metrics. We find that GATA2 protein is expressed in mast cells, subsets of megakaryocytes, CD34-positive stem and progenitor cells (average of 33% in benign and 51% in MDS), and CD117-positive stem and progenitor cells (average of 41% in benign and 40% in MDS). Relatively few Ecad-positive erythroid progenitors co-express GATA2 protein (average of 5% in benign and 8% in MDS). MPO and GATA2 co-expression was not observed. The percent of GATA2-positive cells strongly correlated with the bone marrow blast count across 20 MDS cases (p< 0.001). Follow up anti-GATA2 flow cytometric studies will more precisely map the pattern of GATA2 protein expression within the CD34-positive compartment in benign and MDS bone marrow from human patients. GATA binding factor 2 (GATA2) is a zinc finger transcription factor that regulates complex genetic programs to promote development of the hematopoietic system in the embryo and proper function of the bone marrow in adults. GATA2 is upregulated at the RNA transcript level in myelodysplastic syndrome (MDS), a heterogeneous group of clonal hematopoietic stem cell malignancies characterized by peripheral cytopenias, ineffective hematopoiesis, genetic mutations, and a predisposition to develop acute myeloid leukemia (AML). We sought to directly visualize GATA2 protein expression in benign and MDS bone marrow core biopsies from human patients to identify the cellular compartments that express GATA2 protein and to compare GATA2 protein expression in benign bone marrows to bone marrows involved by MDS. We optimized an affinity purified polyclonal rabbit anti-GATA2 antibody for immunohistochemistry (IHC) on decalcified human patient bone marrow core biopsies and established dual-IHC protocols for GATA2 and CD34, CD117/c-Kit (CD117), E-Cadherin (Ecad), and Myeloperoxidase (MPO). GATA2 expression was then scored and compared to clinicopathologic metrics. We find that GATA2 protein is expressed in mast cells, subsets of megakaryocytes, CD34-positive stem and progenitor cells (average of 33% in benign and 51% in MDS), and CD117-positive stem and progenitor cells (average of 41% in benign and 40% in MDS). Relatively few Ecad-positive erythroid progenitors co-express GATA2 protein (average of 5% in benign and 8% in MDS). MPO and GATA2 co-expression was not observed. The percent of GATA2-positive cells strongly correlated with the bone marrow blast count across 20 MDS cases (p< 0.001). Follow up anti-GATA2 flow cytometric studies will more precisely map the pattern of GATA2 protein expression within the CD34-positive compartment in benign and MDS bone marrow from human patients.

We report a case of thrombotic microangiopathy (TMA) diagnosed in the kidney allograft of a 59-year-old woman who had developed plasmacytoma type monomorphic posttransplant lymphoproliferative disorder (PTLD) 30 years after a simultaneous pancreas and kidney transplant. This report demonstrates a rare etiology of TMA in a kidney allograft occurring in association with PTLD-associated monoclonal gammopathy. The main teaching points are: 1) PTLD may be considered in the work-up of a new TMA in the immunosuppressed post-transplant setting, and 2) a paraprotein-associated condition should be considered as clinically relevant coexistent in a patient with TMA because paraproteins are over-represented in patients with otherwise unexplained TMAs.

Myeloproliferative neoplasms (MPNs) transform to myelofibrosis (MF) and highly lethal acute myeloid leukemia (AML), although the actionable mechanisms driving progression remain elusive. Here, we elucidate the role of the high mobility group A1 (HMGA1) chromatin regulator as a novel driver of MPN progression. HMGA1 is upregulated in MPN, with highest levels after transformation to MF or AML. To define HMGA1 function, we disrupted gene expression via CRISPR/Cas9, short hairpin RNA, or genetic deletion in MPN models. HMGA1 depletion in JAK2V617F AML cell lines disrupts proliferation, clonogenicity, and leukemic engraftment. Surprisingly, loss of just a single Hmga1 allele prevents progression to MF in JAK2V617F mice, decreasing erythrocytosis, thrombocytosis, megakaryocyte hyperplasia, and expansion of stem and progenitors, while preventing splenomegaly and fibrosis within the spleen and BM. RNA-sequencing and chromatin immunoprecipitation sequencing revealed HMGA1 transcriptional networks and chromatin occupancy at genes that govern proliferation (E2F, G2M, mitotic spindle) and cell fate, including the GATA2 master regulatory gene. Silencing GATA2 recapitulates most phenotypes observed with HMGA1 depletion, whereas GATA2 re-expression partially rescues leukemogenesis. HMGA1 transactivates GATA2 through sequences near the developmental enhancer (+9.5), increasing chromatin accessibility and recruiting active histone marks. Further, HMGA1 transcriptional networks, including proliferation pathways and GATA2, are activated in human MF and MPN leukemic transformation. Importantly, HMGA1 depletion enhances responses to the JAK2 inhibitor, ruxolitinib, preventing MF and prolonging survival in murine models of JAK2V617F AML. These findings illuminate HMGA1 as a key epigenetic switch involved in MPN transformation and a promising therapeutic target to treat or prevent disease progression.

Germline coding and enhancer variants within the hematopoietic regulator GATA2 create a bone marrow failure and leukemia predisposition (Soukup and Bresnick, 2020). The conserved murine enhancer (+9.5) promotes hematopoietic stem cell (HSC) genesis, and a single-nucleotide human variant in an Ets motif attenuates chemotherapy- and transplantation-induced hematopoietic regeneration (Soukup et al., 2019), response to inflammatory stresses, and stem and progenitor cell mobilization with the therapeutic mobilizer G-CSF (Soukup et al., 2021). We tested whether +9.5 mutation universally impairs mobilization in response to different stimuli, or if selected pathways are impacted. While treatment with the CXCL2 agonist IL-8 or VLA-4 antagonist BIO5192 resulted in 3.9- and 2.2-fold increased wild type (WT) colony forming units (CFU), no significant increases were detected with mutants. By contrast, mobilization with the clinically utilized CXCR4 antagonist plerixafor resulted in a 4.3-fold increase in WT CFU (P < 0.0001) and a 4.5-fold increase in mutant CFU (P = 0.004). Thus, +9.5 mutation discriminately attenuated stem and progenitor cell mobilization through select, but not all, mechanisms. In addition to G-CSF-induced signaling and HSPC mobilization, we identified two additional mobilization axes (IL-8/CXCL2 and VLA-4/VCAM1) sensitive to genetic variation in the GATA2 enhancer. By contrast, mobilization through the CXCR4/CXCL12 axis was insensitive to the genetic variation. These studies demonstrate how genetic variation in non-coding elements dictates the efficacy of HSPC mobilization. The differential impact of variation on distinct mobilization regimens provides a unique foundation for elucidating how GATA2 and its complex genetic, protein and small molecule networks function as vital components of HSPC-mobilizing mechanisms. Germline coding and enhancer variants within the hematopoietic regulator GATA2 create a bone marrow failure and leukemia predisposition (Soukup and Bresnick, 2020). The conserved murine enhancer (+9.5) promotes hematopoietic stem cell (HSC) genesis, and a single-nucleotide human variant in an Ets motif attenuates chemotherapy- and transplantation-induced hematopoietic regeneration (Soukup et al., 2019), response to inflammatory stresses, and stem and progenitor cell mobilization with the therapeutic mobilizer G-CSF (Soukup et al., 2021). We tested whether +9.5 mutation universally impairs mobilization in response to different stimuli, or if selected pathways are impacted. While treatment with the CXCL2 agonist IL-8 or VLA-4 antagonist BIO5192 resulted in 3.9- and 2.2-fold increased wild type (WT) colony forming units (CFU), no significant increases were detected with mutants. By contrast, mobilization with the clinically utilized CXCR4 antagonist plerixafor resulted in a 4.3-fold increase in WT CFU (P < 0.0001) and a 4.5-fold increase in mutant CFU (P = 0.004). Thus, +9.5 mutation discriminately attenuated stem and progenitor cell mobilization through select, but not all, mechanisms. In addition to G-CSF-induced signaling and HSPC mobilization, we identified two additional mobilization axes (IL-8/CXCL2 and VLA-4/VCAM1) sensitive to genetic variation in the GATA2 enhancer. By contrast, mobilization through the CXCR4/CXCL12 axis was insensitive to the genetic variation. These studies demonstrate how genetic variation in non-coding elements dictates the efficacy of HSPC mobilization. The differential impact of variation on distinct mobilization regimens provides a unique foundation for elucidating how GATA2 and its complex genetic, protein and small molecule networks function as vital components of HSPC-mobilizing mechanisms.