Caspase 8 deletion causes infection/inflammation-induced bone marrow failure and MDS-like disease in mice.

Myelodysplastic syndromes (MDS) are a heterogeneous group of pre-leukemic hematopoietic disorders characterized by cytopenia in peripheral blood due to ineffective hematopoiesis and normo- or hypercellularity and morphologic dysplasia in bone marrow (BM). An inflammatory BM microenvironment and programmed cell death of hematopoietic stem/progenitor cells (HSPCs) are thought to be the major causes of ineffective hematopoiesis in MDS. Pyroptosis, apoptosis and necroptosis (collectively, PANoptosis) are observed in BM tissues of MDS patients, suggesting an important role of PANoptosis in MDS pathogenesis. Caspase 8 (Casp8) is a master regulator of PANoptosis, which is downregulated in HSPCs from most MDS patients and abnormally spliced in HSPCs from MDS patients with SRSF2 mutation. To study the role of PANoptosis in hematopoiesis, we generated inducible Casp8 knockout mice (Casp8-/-). Mx1-Cre-Casp8-/- mice died of BM failure within 10 days of polyI:C injections due to depletion of HSPCs. Rosa-ERT2Cre-Casp8-/- mice are healthy without significant changes in BM hematopoiesis within the first 1.5 months after Casp8 deletion. Such mice developed BM failure upon infection or low dose polyI:C/LPS injections due to the hypersensitivity of Casp8-/- HSPCs to infection or inflammation-induced necroptosis which can be prevented by Ripk3 deletion. However, impaired self-renewal capacity of Casp8-/- HSPCs cannot be rescued by Ripk3 deletion due to activation of Ripk1-Tbk1 signaling. Most importantly, mice transplanted with Casp8-/- BM cells developed MDS-like disease within 4 months of transplantation as demonstrated by anemia, thrombocytopenia and myelodysplasia. Our study suggests an essential role for a balance in Casp8, Ripk3-Mlkl and Ripk1-Tbk1 activities in the regulation of survival and self-renewal of HSPCs, the disruption of which induces inflammation and BM failure, resulting in MDS-like disease.

Role of TET dioxygenases in the regulation of both normal and pathological hematopoiesis.

The family of ten-eleven translocation dioxygenases (TETs) consists of TET1, TET2, and TET3. Although all TETs are expressed in hematopoietic tissues, only TET2 is commonly found to be mutated in age-related clonal hematopoiesis and hematopoietic malignancies. TET2 mutation causes abnormal epigenetic landscape changes and results in multiple stages of lineage commitment/differentiation defects as well as genetic instability in hematopoietic stem/progenitor cells (HSPCs). TET2 mutations are founder mutations (first hits) in approximately 40-50% of cases of TET2-mutant (TET2MT) hematopoietic malignancies and are later hits in the remaining cases. In both situations, TET2MT collaborates with co-occurring mutations to promote malignant transformation. In TET2MT tumor cells, TET1 and TET3 partially compensate for TET2 activity and contribute to the pathogenesis of TET2MT hematopoietic malignancies. Here we summarize the most recent research on TETs in regulating of both normal and pathogenic hematopoiesis. We review the concomitant mutations and aberrant signals in TET2MT malignancies. We also discuss the molecular mechanisms by which concomitant mutations and aberrant signals determine lineage commitment in HSPCs and the identity of hematopoietic malignancies. Finally, we discuss potential strategies to treat TET2MT hematopoietic malignancies, including reverting the methylation state of TET2 target genes and targeting the concomitant mutations and aberrant signals.

Isolated leukemia cutis as extramedullary relapse of acute myeloid leukemia.

Acute myeloid leukemia (AML) arises from clonal expansion of malignant hematopoietic precursor cells in the bone marrow. In rare instances, AML can recur with prominent extramedullary manifestations (i.e., leukemia cutis or myeloid sarcoma), either simultaneously or preceding marrow involvement, or as a sole site of primary disease relapse.

A Shift in Myeloid Cell Phenotype via Down Regulation of Siglec-1 in Island Macrophages of Bone Marrow Is Associated With Decreased Late Erythroblasts Seen in Anemia of Critical Illness.

Burn injury has been shown to significantly dampen erythropoiesis in both burn patients and in murine models. Our previous findings elucidated the erythropoietin independent defects in red cell development stages involving erythroid progenitor production and late stage erythroblast enucleation processes. We hypothesized that macrophages (MØ) in erythroblast islands (EBI) could be yet another roadblock impeding erythropoiesis following burn injury. Here we highlight that the methodology to study EBI can be achieved with single cell suspensions using a simple technique such as flow cytometry, as obtaining the central erythroblast island macrophages (EBIMØs) of interest is a delicate process. We elucidated the requisite of EBIMØ from the erythroblast as well as the MØ perspective. In addition to the primary erythropoiesis organ, the bone marrow (BM), spleens were also examined for extra-medullary erythropoiesis. Femurs and spleens were harvested from adult mice (B6D2F1) subjected to 15% total body surface area (TBSA) scald burn (B) or sham burn (S). Total bone marrow cells (TBM) and splenocytes were probed for total erythrons, early and late erythroblasts and EBIMØ by flow cytometry. There was only a marginal increase in the number of EBIMØ after burn, but, between the signatures of EBIMØ, Siglec-1 expression (MFI) was reduced by 40% in B with and a parallel 44% decrease in TBM erythrons in the BM. There were more (2.5-fold) EEBs and less LEBs (2.4-fold) per million TBM cells in B; with a corresponding decrease in Siglec-1 and Ly6G expressions in EBIMØ associated with EEB. Conversely, extra-medullary erythropoiesis was robust in spleens from B. Not only were the numbers of EBIMØs increased in B (p < 0.002), both EEBs and LEBs associated with EBIMØ were higher by 30 and 75%, respectively. Importantly, an increase in Siglec-1 and Vcam1 expressing F480+ splenic macrophages was observed after burn injury. Therefore, stagnant EEBs in the BM after burn injury could be due to low Siglec1 expressing EBIMØ, which perhaps impede their maturation into LEBs and reticulocytes. Repercussion of myeloid cell phenotype specific to BM after burn injury could plausibly account for a defective late stage RBC maturation resulting in anemia of critical illness. Summary Sentence: Characterization of erythroblast island macrophages (EBIMØ) in the bone marrow and spleen at different stages of erythropoiesis after burn injury.

Self-assembling vascular endothelial growth factor nanoparticles improve function in spinocerebellar ataxia type 1.

There is increasing appreciation for the role of the neurovascular unit in neurodegenerative diseases. We showed previously that the angiogenic and neurotrophic cytokine, vascular endothelial growth factor (VEGF), is suppressed to abnormally low levels in spinocerebellar ataxia type 1 (SCA1), and that replenishing VEGF reverses the cerebellar pathology in SCA1 mice. In that study, however, we used a recombinant VEGF, which is extremely costly to manufacture and biologically unstable as well as immunogenic. To develop a more viable therapy, here we test a synthetic VEGF peptide amphiphile that self-assembles into nanoparticles. We show that this nano-VEGF has potent neurotrophic and angiogenic properties, is well-tolerated, and leads to functional improvement in SCA1 mice even when administered at advanced stages of the disease. This approach can be generalized to other neurotrophic factors or molecules that act in a paracrine manner, offering a novel therapeutic strategy for neurodegenerative conditions.

Necroptosis in spontaneously-mutated hematopoietic cells induces autoimmune bone marrow failure in mice.

Acquired aplastic anemia is an autoimmune-mediated bone marrow failure syndrome. The mechanism by which such an autoimmune reaction is initiated is unknown. Whether and how the genetic lesions detected in patients cause autoimmune bone marrow failure have not yet been determined. We found that mice with spontaneous deletion of the TGFß-activated kinase-1 gene in a small subset of hematopoietic cells developed bone marrow failure which resembled the clinical manifestations of acquired aplastic anemia patients. Bone marrow failure in such mice could be reversed by depletion of CD4+ T lymphocytes or blocked by knockout of interferon-γ, suggesting a Th1-cell-mediated autoimmune mechanism. The onset and progression of bone marrow failure in such mice were significantly accelerated by the inactivation of tumor necrosis factor-α signaling. Tumor necrosis factor-α restricts autoimmune bone marrow failure by inhibiting type-1 T-cell responses and maintaining the function of myeloid-derived suppressor cells. Furthermore, we determined that necroptosis among a small subset of mutant hematopoietic cells is the cause of autoimmune bone marrow failure because such bone marrow failure can be prevented by deletion of receptor interacting protein kinase-3 Our study suggests a novel mechanism to explain the pathogenesis of autoimmune bone marrow failure.

The role of the Exon 13 G571S JAK2 mutation in myeloproliferative neoplasms.

The exon 14 JAK2 V617F mutation has been well established as a driver mutation in polycythemia vera (PV) and other myeloproliferative neoplasms. JAK2 exon 12 mutations have also been implicated in PV, although patients with these mutations may show isolated erythrocytosis. Recently additional JAK2point mutations have been described-all in regions encoding the pseudokinase domain that regulates the tyrosine kinase activity of JAK2. We present a case of a patient with erythrocytosis and an exon 13 G571S mutation, and discuss the putative role of this mutation in myeloproliferative neoplasms.

FAK mediates a compensatory survival signal parallel to PI3K-AKT in PTEN-null T-ALL cells.

Mutations and inactivation of phosphatase and tensin homolog deleted from chromosome 10 (PTEN) are observed in 15%-25% of cases of human T cell acute lymphoblastic leukemia (T-ALL). Pten deletion induces myeloproliferative disorders (MPDs), acute myeloid leukemia (AML), and/or T-ALL in mice. Previous studies attributed Pten-loss-related hematopoietic defects and leukemogenesis to excessive activation of phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signaling. Although inhibition of this signal dramatically suppresses the growth of PTEN-null T-ALL cells in vitro, treatment with inhibitors of this pathway does not cause a complete remission in vivo. Here, we report that focal adhesion kinase (Fak), a protein substrate of Pten, also contributes to T-ALL development in Pten-null mice. Inactivation of the FAK signaling pathway by either genetic or pharmacologic methods significantly sensitizes both murine and human PTEN-null T-ALL cells to PI3K/AKT/mTOR inhibition when cultured in vitro on feeder layer cells or a matrix and in vivo.

Anaplastic large cell lymphoma involving the urinary bladder: a case report and review of the literature.

T cell-derived malignant lymphoma is rarely detected as a bladder neoplasm. A literature review for anaplastic large cell lymphoma (ALCL) involving urinary bladder reveals only seven previously reported cases. Here, we report a case of a 59-year-old HIV-negative man with ALK-positive ALCL. He presented an unusual clinical course with initial consideration of adult onset Still's Disease (AOSD) due to his negative results searching for malignancy and infectious diseases. He rapidly developed macrophage activation (hemophagocytic) syndrome and experienced an unusual rapid disease progression and died in 39 days after onset of symptoms. Compared to previously reported cases, the current case of ALK-1-positive ALCL is a rare case with an unusual presentation. From this case, we learned that ALCL is one malignancy that should be considered and screened in patients with suspected AOSD. Also, T-cell lymphoma associated hemophagocytic syndrome should be considered in a patient with sustained corticosteroid-resistant spike fever, high serum ferritin, and rapid exacerbation of the disease course.

Analysis of immunoglobulin heavy chain rearrangement in a child with recurrent Burkitt lymphoma to determine clonality.

BACKGROUND: Burkitt lymphoma (BL) is an aggressive, rapidly proliferating neoplasm of B-cell origin. A late recurrence should be investigated to differentiate whether it is a true relapse or a de novo lymphoma as this has therapeutic implications. OBSERVATIONS: We report an HIV-negative white male individual, who at the age of 14 presented with recurrent BL in the abdomen occurring 6 years after successful treatment. Analysis of VDJ rearrangement showed marked dissimilarity in clonal peaks between the 2 tumors, suggesting that each tumor was associated with separate origins. The second tumor was treated as a de novo BL, and the patient remained in complete remission 2 years from recurrence without any evidence of the disease. CONCLUSION: We present a case with 2 distinct BLs verified by the VDJ rearrangement analysis in a non-HIV infected individual. Our case supports the finding that a genetically discrete BL can be treated as if it were a new tumor, thereby reducing chemotherapeutic burden and treatment-related morbidity associated with the regimens for relapsed or refractory disease. Analysis of VDJ rearrangement seems to be a reliable assessment of tumor clonality.

Chronic myelomonocytic leukemia monocytes uniformly display a population of monocytes with CD11c underexpression.

OBJECTIVES: To examine the utility of CD11c expression on monocytes in normal controls and patients with chronic myelomonocytic leukemia (CMML) (n = 23) with flow cytometric immunophenotyping. METHODS: Twenty-three CMML samples and 10 control bone marrows submitted for lymphoma staging without evidence of disease were examined. RESULTS: Monocytes in CMML samples ranged from 4% to 35%. Expression of at least one aberrant monocytic marker was found on the monocytes in 18 (82%) of 22 evaluable cases. The most common aberrancy was underexpression of CD11c (n = 15), while none of the bone marrow controls showed underexpression of CD11c. CONCLUSIONS: A distinct heterogeneous population of monocytic cells with underexpression of CD11c was identified in all these cases. CD11c underexpression was independent of other aberrancies, including HLA-DR underexpression (n = 14), aberrant CD56 expression (n = 11), and underexpression of CD33, CD38, and CD14 (n = 6, 5, and 5, respectively), supporting the utility of CD11c expression status on monocytes in establishing a CMML diagnosis.

Burn injury dampens erythroid cell production through reprioritizing bone marrow hematopoietic response.

BACKGROUND: Anemia in burn patients is due to surgical blood loss and anemia of critical illness. Because the commitment paradigm of common bone marrow progenitors dictates the production of erythroid, myeloid, and lymphoid cells, we hypothesized that skewed bone marrow lineage commitment decreases red cell production and causes anemia after a burn injury. METHODS: After anesthesia, B(6)D(2)F(1) mice received a 15% total body surface area dorsal scald burn. The sham group did not receive scald burn. Femoral bone marrow was harvested on 2, 5, 7, 14, and 21 postburn days (PBD). Total bone marrow cells were labeled with specific antibodies to erythroid (CD71/Ter119), myeloid (CD11b), and lymphoid (CD19) lineages and analyzed by flow cytometry. To test whether erythropoietin (EPO) could increase red blood cell production, EPO was administered to sham and burn animals and their reticulocyte response was measured on PBD 2 and PBD 7. RESULTS: Burn injury reduced the erythroid cells of the bone marrow from 35% in sham to 17% by PBD 5 and remained at similar level until PBD 21. Myeloid cells, however, increased from 42% in sham to 60% on PBD 5 and 77% on PBD 21. Burn injury reduced reticulocyte counts on PBD 2 and PBD 7 indicating that the erythroid compartment is severely depleted. This depleted compartment, however, responded to EPO but was not sufficient to change red cell production. CONCLUSION: Burn injury skews the bone marrow hematopoietic commitment away from erythroid and toward myeloid cells. Shrinkage of the erythroid compartment contributes to resistance to EPO and the anemia of critical illness.

Vascular endothelial growth factor ameliorates the ataxic phenotype in a mouse model of spinocerebellar ataxia type 1.

Spinocerebellar ataxia type 1 (SCA1) is an adult-onset, dominantly inherited neurodegenerative disease caused by expansion of a glutamine repeat tract in ataxin-1 (ATXN1). Although the precise function of ATXN1 remains elusive, it seems to be involved in transcriptional repression. We find that mutant ATXN1 represses transcription of the neurotrophic and angiogenic factor vascular endothelial growth factor (VEGF). Genetic overexpression or pharmacologic infusion of recombinant VEGF mitigates SCA1 pathogenesis, suggesting a new therapeutic strategy for this disease.

c-Myc-mediated control of cell fate in megakaryocyte-erythrocyte progenitors.

It has been found that c-Myc protein plays a critical role in controlling self-renewal versus differentiation in hematopoietic stem cells. We report that c-Myc also controls the fate of megakaryocyte-erythrocyte progenitors through regulating the differentiation of erythroid and megakaryocytic progenitors. In addition to the significant reduction of granulocytes/macrophages and B and T lymphocytes because of the reduction of their corresponding progenitors, we found significantly increased numbers of megakaryocytic progenitors and mature megakaryocytes in bone marrow and spleens of c-Myc-knockout (c-Myc(-/-)) mice. Differentiation of erythrocytes was blocked at the erythroid progenitor stage. This increased megakaryocytopoiesis is a cell-intrinsic defect of c-Myc-mutant hematopoietic stem cells, as shown by transplantation studies. Furthermore, we found that c-Myc is required for polyploidy formation but not for cytoplasmic maturation of megakaryocytes. Megakaryocytes from c-Myc(-/-) mice are significantly smaller in size and lower in ploidy than those of control mice; however, because of the dramatic increase in megakaryocyte number, although fewer platelets are produced by each megakaryocyte, a greater than 3-fold increase in platelet number was consistently observed in c-Myc(-/-) mice. Thus, c-Myc(-/-) mice develop a syndrome of severe thrombocytosis-anemia-leukopenia because of significant increases in megakaryocytopoiesis and concomitant blockage of erythrocyte differentiation and reductions in myelolymphopoiesis.

Neuronal differentiation is regulated by leucine-rich acidic nuclear protein (LANP), a member of the inhibitor of histone acetyltransferase complex.

Neuronal differentiation is a tightly regulated process characterized by temporal and spatial alterations in gene expression. A number of studies indicate a significant role for histone acetylation in the regulation of genes involved in development. Histone acetylation is regulated by histone deacetylases and histone acetyltransferases. Recent findings suggest that these catalytic activities, in turn, are modulated by yet another set of regulators. Of considerable interest in this context is the possible role of the INHAT (inhibitor of histone acetyltransferase) complex, comprised of a group of acidic proteins that suppress histone acetylation by a novel "histone-masking" mechanism. In this study, we specifically examined the role of the leucine-rich acidic nuclear protein (LANP), a defining member of the INHAT complex whose expression is tightly regulated in neuronal development. We report that depleting LANP in neuronal cell lines promotes neurite outgrowth by inducing changes in gene expression. In addition, we show that LANP directly regulates expression of the neurofilament light chain, an important neuron-specific cytoskeletal gene, by binding to the promoter of this gene and modulating histone acetylation levels. Finally, we corroborated our findings in vivo by demonstrating increased neurite outgrowth in primary neurons obtained from LANP null mice, which is also accompanied by increased histone acetylation at the NF-L promoter. Taken together, these results implicate INHATs as a distinct class of developmental regulators involved in the epigenetic modulation of neuronal differentiation.

HSP90 inhibitor 17AAG causes apoptosis in ATRA-resistant acute promyelocytic leukemia cells.

The effects of a novel heat shock protein inhibitor, 17AAG, on established APL cell lines (NB4 and R1) were analyzed. 17AAG induces apoptosis in APL cell lines both sensitive (NB4) and resistant (R1) to ATRA after 72 h of incubation. Apoptosis occurs by a mechanism different than ATRA-mediated response, as the cells do not undergo differentiation before apoptosis. Analysis of bax and bcl-2 shows that pro-apoptotic (bax) and anti-apoptotic (bcl-2) proteins are decreased in expression after incubation with 17AAG. We believe this data supports potential clinical use of agents that target HSP90 in APL patients failing conventional therapy.