Linking GATA2 to myeloid dysplasia and complex cytogenetics in adult myelodysplastic neoplasm and acute myeloid leukemia.

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.

Diffuse Large B-Cell Lymphoma, Not Otherwise Specified (DLBCL NOS) Presenting as Multiple Subcutaneous Nodules: An Unusual Cutaneous Presentation of Systemic Disease.

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.

A gain-of-function p53 mutant synergizes with oncogenic NRAS to promote acute myeloid leukemia in mice.

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.

Spatial transcriptomics of a giant pilomatricoma.

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.

Needle in a haystack or elephant in the room? Identifying germline predisposition syndromes in the setting of a new myeloid malignancy diagnosis.

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.

Aurora A phosphorylates Ndel1 to reduce the levels of Mad1 and NuMA at spindle poles.

Dynein inactivates the spindle assembly checkpoint (SAC) by transporting checkpoint proteins away from kinetochores toward spindle poles in a process known as "stripping." 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 absent. 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 nonphosphorylatable mutant in human cells. The pole focusing protein NuMA, transported to poles by dynein, also accumulates at poles in cells harboring a mutant Ndel1. Phosphorylation 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.

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

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.

A "Null" Pattern of p16 Immunostaining in Endometrial Serous Carcinoma: An Under-recognized and Important Aberrant Staining Pattern.

The ability to distinguish endometrial serous carcinoma (SC) from high-grade endometrioid adenocarcinoma is of great importance given their differences in prognosis and management. In practice, this distinction typically relies upon the use of a focused immunohistochemical panel including p53, p16, and mismatch repair proteins. The expression of p16 is characteristically strong and diffuse in SCs, and weak and/or patchy in many high-grade endometrioid adenocarcinomas. Here, we report a subset of SCs that are entirely negative for p16 immunostaining, a pattern we refer to as "p16 null." This pattern was identified in 2 of 63 cases of SC diagnosed at our institution-1 with histologically classic features and 1 with ambiguous high-grade histologic features. These tumors otherwise showed a SC signature by immunohistochemical and demonstrated an SC pattern of genetic mutations. No mutation in the gene for p16, cyclin-dependent kinase inhibitor 2A (CDKN2A), was identified in either case. However, molecular correlates for the absent p16 expression were present, including homozygous deletion of CDKN2A in one case and hemizygous deletion of CDKN2A with promotor hypermethylation of the remaining allele in the other case. To our knowledge, this constitutes the first report conclusively demonstrating the existence of a small subset of SCs that are completely negative by p16 immunohistochemistry, and the molecular lesions responsible for this pattern. In the context of an otherwise clinically and histologically classic example of SC, we endorse this "null" p16 staining pattern as an alternative aberrant staining pattern that should not deter one from committing to this diagnosis.

Conditionally pathogenic genetic variants of a hematopoietic disease-suppressing enhancer.

Human genetic variants are classified on the basis of potential pathogenicity to guide clinical decisions. However, mechanistic uncertainties often preclude definitive categorization. Germline coding and enhancer variants within the hematopoietic regulator GATA2 create a bone marrow failure and leukemia predisposition. The conserved murine enhancer promotes hematopoietic stem cell (HSC) genesis, and a single-nucleotide human variant in an Ets motif attenuates chemotherapy-induced hematopoietic regeneration. We describe "conditionally pathogenic" (CP) enhancer motif variants that differentially affect hematopoietic development and regeneration. The Ets motif variant functioned autonomously in hematopoietic cells to disrupt hematopoiesis. Because an epigenetically silenced normal allele can exacerbate phenotypes of a pathogenic heterozygous variant, we engineered a bone marrow failure model harboring the Ets motif variant and a severe enhancer mutation on the second allele. Despite normal developmental hematopoiesis, regeneration in response to chemotherapy, inflammation, and a therapeutic HSC mobilizer was compromised. The CP paradigm informs mechanisms underlying phenotypic plasticity and clinical genetics.

RNA-regulatory exosome complex confers cellular survival to promote erythropoiesis.

Cellular differentiation requires vast remodeling of transcriptomes, and therefore machinery mediating remodeling controls differentiation. Relative to transcriptional mechanisms governing differentiation, post-transcriptional processes are less well understood. As an important post-transcriptional determinant of transcriptomes, the RNA exosome complex (EC) mediates processing and/or degradation of select RNAs. During erythropoiesis, the erythroid transcription factor GATA1 represses EC subunit genes. Depleting EC structural subunits prior to GATA1-mediated repression is deleterious to erythroid progenitor cells. To assess the importance of the EC catalytic subunits Dis3 and Exosc10 in this dynamic process, we asked if these subunits function non-redundantly to control erythropoiesis. Dis3 or Exosc10 depletion in primary murine hematopoietic progenitor cells reduced erythroid progenitors and their progeny, while sparing myeloid cells. Dis3 loss severely compromised erythroid progenitor and erythroblast survival, rendered erythroblasts hypersensitive to apoptosis-inducing stimuli and induced γ-H2AX, indicative of DNA double-stranded breaks. Dis3 loss-of-function phenotypes were more severe than those caused by Exosc10 depletion. We innovated a genetic rescue system to compare human Dis3 with multiple myeloma-associated Dis3 mutants S447R and R750K, and only wild type Dis3 was competent to rescue progenitors. Thus, Dis3 establishes a disease mutation-sensitive, cell type-specific survival mechanism to enable a differentiation program.

Selective Requirement of MYB for Oncogenic Hyperactivation of a Translocated Enhancer in Leukemia.

In acute myeloid leukemia (AML) with inv(3)(q21;q26) or t(3;3)(q21;q26), a translocated GATA2 enhancer drives oncogenic expression of EVI1. We generated an EVI1-GFP AML model and applied an unbiased CRISPR/Cas9 enhancer scan to uncover sequence motifs essential for EVI1 transcription. Using this approach, we pinpointed a single regulatory element in the translocated GATA2 enhancer that is critically required for aberrant EVI1 expression. This element contained a DNA-binding motif for the transcription factor MYB, which specifically occupied this site at the translocated allele and was dispensable for GATA2 expression. MYB knockout as well as peptidomimetic blockade of CBP/p300-dependent MYB functions resulted in downregulation of EVI1 but not of GATA2. Targeting MYB or mutating its DNA-binding motif within the GATA2 enhancer resulted in myeloid differentiation and cell death, suggesting that interference with MYB-driven EVI1 transcription provides a potential entry point for therapy of inv(3)/t(3;3) AMLs. SIGNIFICANCE: We show a novel paradigm in which chromosomal aberrations reveal critical regulatory elements that are nonfunctional at their endogenous locus. This knowledge provides a rationale to develop new compounds to selectively interfere with oncogenic enhancer activity.This article is highlighted in the In This Issue feature, p. 2659.

Concurrent Central Diabetes Insipidus and Acute Myeloid Leukemia.

Central diabetes insipidus (CDI) is a rare reported complication of acute myeloid leukemia (AML). The onset of AML-associated CDI often precedes the diagnosis of AML by weeks or months and is considered an adverse prognostic indicator in this setting. The mechanism of AML-associated CDI is not known; however, it is often reported in the setting of cytogenetic events resulting in MDS1 and EVI1 complex locus protein (MECOM) gene overexpression. Here, we describe a case of new onset CDI which preceded a diagnosis of AML by 1 month. We detail the clinical and laboratory evaluation of the patient's CDI, and we describe the pathological and laboratory workup of their AML, which ultimately yielded a diagnosis of AML with myelodysplasia-related changes. Additional cytogenetic findings included the identification of the t (2;3)(p23;q27), which leads to MECOM gene overexpression and which to our knowledge has not previously been reported in the setting of AML-associated CDI. The patient received induction chemotherapy followed by allogeneic hematopoietic stem cell transplantation but experienced disease relapse and passed away nine months after initial diagnosis. We emphasize that new onset CDI can occur as a rare complication of AML where it portends a poor prognosis and may be related to AML subtypes displaying MECOM gene dysregulation.

High nuclear TPX2 expression correlates with TP53 mutation and poor clinical behavior in a large breast cancer cohort, but is not an independent predictor of chromosomal instability.

BACKGROUND: Targeting Protein for Xenopus Kinesin Like Protein 2 (TPX2) is a microtubule associated protein that functions in mitotic spindle assembly. TPX2 also localizes to the nucleus where it functions in DNA damage repair during S-phase. We and others have previously shown that TPX2 RNA levels are strongly associated with chromosomal instability (CIN) in breast and other cancers, and TPX2 RNA levels have been demonstrated to correlate with aggressive behavior and poor clinical outcome across a range of solid malignancies, including breast cancer. METHODS: We perform TPX2 IHC on a cohort of 253 primary breast cancers and adopt a clinically amenable scoring system to separate tumors into low, intermediate, or high TPX2 expression. We then correlate TPX2 expression against diverse pathologic parameters and important measures of clinical outcome, including disease-specific and overall survival. We link TPX2 expression to TP53 mutation and evaluate whether TPX2 is an independent predictor of chromosomal instability (CIN). RESULTS: We find that TPX2 nuclear expression strongly correlates with high grade morphology, elevated clinical stage, negative ER and PR status, and both disease-specific and overall survival. We also show that increased TPX2 nuclear expression correlates with elevated ploidy, supernumerary centrosomes, and TP53 mutation. TPX2 nuclear expression correlates with CIN via univariate analyses but is not independently predictive when compared to ploidy, Ki67, TP53 mutational status, centrosome number, and patient age. CONCLUSIONS: Our findings demonstrate a strong correlation between TPX2 nuclear expression and aggressive tumor behavior, and show that TPX2 overexpression frequently occurs in the setting of TP53 mutation and elevated ploidy. However, TPX2 expression is not an independent predictor of CIN where it fails to outperform existing clinical and pathologic metrics.

Sterile α-motif domain requirement for cellular signaling and survival.

Hundreds of sterile α-motif (SAM) domains have predicted structural similarities and are reported to bind proteins, lipids, or RNAs. However, the majority of these domains have not been analyzed functionally. Previously, we demonstrated that a SAM domain-containing protein, SAMD14, promotes SCF/proto-oncogene c-Kit (c-Kit) signaling, erythroid progenitor function, and erythrocyte regeneration. Deletion of a Samd14 enhancer (Samd14-Enh), occupied by GATA2 and SCL/TAL1 transcription factors, reduces SAMD14 expression in bone marrow and spleen and is lethal in a hemolytic anemia mouse model. To rigorously establish whether Samd14-Enh deletion reduces anemia-dependent c-Kit signaling by lowering SAMD14 levels, we developed a genetic rescue assay in murine Samd14-Enh-/- primary erythroid precursor cells. SAMD14 expression at endogenous levels rescued c-Kit signaling. The conserved SAM domain was required for SAMD14 to increase colony-forming activity, c-Kit signaling, and progenitor survival. To elucidate the molecular determinants of SAM domain function in SAMD14, we substituted its SAM domain with distinct SAM domains predicted to be structurally similar. The chimeras were less effective than SAMD14 itself in rescuing signaling, survival, and colony-forming activities. Thus, the SAMD14 SAM domain has attributes that are distinct from other SAM domains and underlie SAMD14 function as a regulator of cellular signaling and erythrocyte regeneration.

Autoimmune Lymphoproliferative Syndrome: An Overview.

Autoimmune lymphoproliferative syndrome (ALPS) is an inherited nonmalignant lymphoproliferative disorder characterized by heterozygous mutations within the first apoptosis signal receptor (FAS) signaling pathway. Defects in FAS-mediated apoptosis cause an expansion and accumulation of autoreactive CD4- and CD8- (double-negative) T cells, leading to cytopenias, splenomegaly, lymphadenopathy, autoimmune disorders, and a greatly increased lifetime risk of lymphoma. The differential diagnosis of ALPS includes infection, other inherited immunodeficiency disorders, primary and secondary autoimmune syndromes, and lymphoma. The most consistent pathologic feature is a florid paracortical expansion of double-negative T cells in lymph nodes. A presumptive clinical diagnosis can be made from symptoms and a constellation of laboratory test results. However, a definitive diagnosis requires ancillary testing and enables disease subclassification. Recognition of ALPS is critical, as treatment with immunosuppressive therapies can effectively reduce or ameliorate symptoms for most patients.

GATA2 Promotes Hematopoietic Development and Represses Cardiac Differentiation of Human Mesoderm.

In vertebrates, GATA2 is a master regulator of hematopoiesis and is expressed throughout embryo development and in adult life. Although the essential role of GATA2 in mouse hematopoiesis is well established, its involvement during early human hematopoietic development is not clear. By combining time-controlled overexpression of GATA2 with genetic knockout experiments, we found that GATA2, at the mesoderm specification stage, promotes the generation of hemogenic endothelial progenitors and their further differentiation to hematopoietic progenitor cells, and negatively regulates cardiac differentiation. Surprisingly, genome-wide transcriptional and chromatin immunoprecipitation analysis showed that GATA2 bound to regulatory regions, and repressed the expression of cardiac development-related genes. Moreover, genes important for hematopoietic differentiation were upregulated by GATA2 in a mostly indirect manner. Collectively, our data reveal a hitherto unrecognized role of GATA2 as a repressor of cardiac fates, and highlight the importance of coordinating the specification and repression of alternative cell fates.

Association of Immune Thrombocytopenia and T-Lymphoblastic Lymphoma in a Pediatric Patient.

Immune thrombocytopenia (ITP) is characterized by isolated thrombocytopenia of unclear etiology. We present a unique case of an 8-year-old girl with chronic ITP who was subsequently diagnosed with T-lymphoblastic lymphoma at age 11. The clinical course was complicated by the occurrence of nonepileptiform events with bizarre behavior changes following the administration of nelarabine and intrathecal and high-dose systemic methotrexate. This case highlights an unusual co-occurrence of hematologic malignancy and chronic ITP in an otherwise healthy child. We speculate that underlying genetic or immunologic lesions may predispose a subset of pediatric ITP patients to the development of hematologic malignancies.

Fatal Myocarditis Following Treatment with the PD-1 Inhibitor Nivolumab.

Therapeutic antibodies targeting the programmed cell death protein 1 (PD-1) pathway function as immune checkpoint inhibitors, allowing the immune system to recognize tumors which otherwise escape immune surveillance. However, these agents can also elicit an autoimmune response by inhibiting the ability of non-neoplastic tissues and regulatory cells to suppress the immune system. Here we present a fatal case of active myocarditis in a 55-year-old man with non-small-cell lung cancer which occurred following monotherapy with the PD-1 inhibitor nivolumab (Opdivo). He presented with acute right-sided heart failure and died 1 day after admission. Postmortem examination revealed multiple gelatinous lesions in the myocardium of the interventricular septum and the bilateral atria and ventricles which had microscopic features diagnostic of myocarditis. Subsequent studies failed to identify an infectious cause. Immune checkpoint inhibitors are an increasingly common addition to anticancer regimens and they should be considered in the evaluation of acute myocarditis.