ALBERT

Description: Cis-regulatory mechanisms control chromatin structure and cellular identity. At the GATA2 locus, two cis-elements are linked to human pathologies, including a primary immunodeficiency (MonoMAC syndrome) associated with multiple complex phenotypes, myelodysplastic syndrome, and acute myeloid leukemia (AML). Mutations that disrupt the function of an intronic GATA2 +9.5 element cause MonoMAC syndrome, while an inversion that relocates the distal GATA2 -77 element to the EVI1 locus induces AML. The +9.5 and -77 cis-elements are GATA-2-occupied and confer context-dependent enhancer activities in select hematopoietic cell types in vivo. In knockout mouse models, the Gata2 +9.5 cis-element is required for hematopoietic stem cell (HSC) genesis, whereas the Gata2 -77 cis-element governs a unique sector of the myeloid progenitor cell transcriptome without impacting HSC genesis. Three other GATA-2-occupied cis-elements (-1.8, -2.8 and -3.9) were not individually required for hematopoietic development, and had relatively mild effects on Gata2 expression; the -1.8 site was required to maintain Gata2 repression in late-stage erythroblasts, the -2.8 conferred maximal Gata2 expression, and the -3.9 had no effect on Gata2 expression. We predict that additional cis-elements exist in the genome with functions resembling the +9.5 and -77, and their analysis will provide important mechanistic and biological insights. We utilized the known properties of Gata2 cis-elements as learning tools to identify prospective constituents of a hematopoietic stem/progenitor cell (HSPC) regulatory cistrome genome-wide. Using sequence attributes shared with the critically-important +9.5 element, namely a CATCTG-8bp spacer-AGATAA, we generated a list of 797 candidate cis-elements ("+9.5-like" elements). This list was prioritized using chromatin occupancy by GATA-2 and Scl/TAL-1, among others, chromatin accessibility, evolutionary conservation, and histone modifications in a multitude of biologically-relevant cell types. Gene editing was used to delete three high-ranked elements (Samd14 +2.5, Bcl2l1 +12.2, and Dapp1 +23.5), revealing their importance for transcriptional activation, GATA-2 occupancy and chromatin accessibility, while deletion of two low-ranked elements (Mrps9 +17.6 and Mgmt +182) had no effect on gene transcription. One such cis-element (Samd14 +2.5) resided in Samd14, a gene with undescribed biological function. Samd14 has a conserved sterile α-motif and coiled-coil domain, and is highly expressed in hematopoietic progenitors and differentiated progeny. Mouse knockout of the Samd14 +2.5 element dramatically lowered expression of Samd14 in hematopoietic progenitors. We conducted loss-of-function analysis to elucidate Samd14 function in lineage-depleted (Lin-) E14.5 fetal liver cells infected with control or Samd14 shRNA-expressing retrovirus. In a CFU assay, Samd14 knockdown reduced BFU-E and CFU-GM colonies 3.4-fold. Early erythroid precursor R1 (CD71low, Ter119-) and R2 (CD71high, Ter119-) cell populations decreased ~2-fold, concomitant with increases in more mature R3 and R4/5 populations (Ter119+). In R1/R2 cells, Samd14 knockdown reduced surface c-Kit expression by 1.6-fold and prevented Stem Cell Factor/c-Kit activation of AKT. Cellular deficits resulting from Samd14 knockdown could be rescued by c-Kit. In -77-/- common myeloid progenitors, Samd14 was ~20-fold downregulated. Thus, the importance of Samd14 and the Samd14 +2.5 element on progenitor function and SCF/c-Kit signaling validates our strategy for identifying cis-elements relevant for hematopoiesis. Our findings demonstrate that +9.5-like elements control cell signaling (Samd14 +2.5) and apoptosis (Bcl2l1 +12.2), and we predict that additional cistrome constituents will control these and other important HSPC processes. I will discuss the mechanistic and biological properties of additional cis-elements analyzed from a cohort of 68 GATA-2-occupied elements and general principles arising from the HSPC cistrome analysis, which provide unique insights into the control of hematopoiesis and GATA-2-linked pathologies. Disclosures No relevant conflicts of interest to declare.

Description: We developed “subfractionation culturing method” for rapid establishment of clonal marrow stomal cell (cMSC) lines. The procedure consists of mixing 1 mL of bone marrow aspirate with 15 mL of complete growth medium (Dulbecco’s Modified Eagle’s Medium containing high glucose, 20% fetal bovine serum [FBS], and 1% penicillin/streptomycin), incubation in a 100 mm culture dish for 2 hours at 37° C with 5% CO2 (I2H), transferring cell culture supernatant a new 100 mm dish, I2H, transferring supernatant to a new dish (D1), I2H, transferring supernatant to a new dish (D2), 1 day incubation (I1D), transferring to another new dish (D3), and I1D in sequence. This process was repeated twice with 1 or 2 day incubation (D4 or D5 respectively). The single-cell derived colonies appearing in the D3, D4 and D5 dishes were transferred to a 6 well plate and then to larger culture flasks, where they kept expanding. After 10 to 14 days in the 100 mm dishes, the cells were harvested with 0.25% trypsin and 1 mM EDTA, suspended at 1 x 106 cells/mL in 10% dimethylsulfoxide and 40% FBS, and frozen in 1-mL aliquots in liquid nitrogen. This method was used for isolation and expansion of cMSCs from the maternal marrow of a 18 year old girl who underwent an allogeneic blood stem cell transplant (BSCT) from unrelated donor for acute biphenotypic leukemia. The isolated cMSCs showed similar characteristics to those of known mesenchymal stem cells in expression of cell surface epitopes and differentiation potentials. For graft versus host disease (GVHD) prophylaxis, methotrexate and cyclosporine were used. She developed de novo chronic GVHD involving eyes, skin, liver, and gut (grade 4 diarrhea) at 7 months postBSCT, a month after withdrawal from GVHD prophylaxis. Intractable bloody diarrhea and malnutrition persisted despite administration of steroid, tacrolimus, and mycophenolate. CMV antigenemia, CMV pancolitis, and hemorrhagic cystitis by BK virus ensued. Jaundice peaked at bilirubin of 4mg/dL. While she was placed on ganciclovir and cidfovir, maternal cMSCs were taken from the bone marrow. At 2 months after onset of GVHD, maternal cMSCs was given twice in 3 week interval, at 2 x 106 cells/kg each, preceded by approval of Inha University IRB as well as Korean FDA. No adverse effect was noted on cMSC infusion. Her diarrhea, malnutrition, skin pigmentation, and ocular sicca got better slowly. Jaundice, CMV antigenemia, and cystitis were gone. At 4 months after onset of GVHD, multiple ulcers in terminal ileum, entire colon, and rectum still remained (proven as GVHD with no evidence of CMV disease). DNA study of biopsied gut revealed triple chimerism. Despite persistent clusters of ulcer at 6 months, the patient did very well in good nutritional status with no diarrhea. She has been off prednisone, given for 12 weeks, and has kept taking tacrolimus, mycophenolate, and trimethoprim/sulfimethoxazole for 9 months. It is unclear how much the cMSCs have contributed to the recovery from GVHD, but the DNA from third party in the gut supports the hypothesis that cMSCs played a role in the repair of damaged gut aside from immune modulatory effect.

Description: The prevalence of ARL is thought to be on increase in Korea. The aim of this study is to review cases of ARL in Korea from August 1998 through July 2006. A total of 16 cases of ARL were reported from 6 institutions in Korea. The patients consisted of 14 males and 2 females at a median age of 41 (range, 30–68) on diagnosis of AIDS. ARL developed at their median age of 43 (range, 32–69). The histologic diagnosis was diffuse large B cell lymphoma (n=9), Burkitt lymphoma (n=1), peripheral T cell lymphoma (n=1), NK/T cell lymphoma (n=1), primary CNS lymphoma (n=1), primary effusion lymphoma (n=1), Hodgkin lymphoma (n=1), or plasmablastic lymphoma (n=1). The disease extent was unknown in 4, stage I in 3, stage II in 4, stage III in 1, stage IV in 5. B symptom was noted in 5 at presentation. Seven of 16 (43.8%) was receiving highly active anti-retroviral therapy (HAART) before the diagnosis of ARL. All the patients were given HAART after the diagnosis of ARL. The response to HARRT was evaluable in 9 patients based on CD4+ cell count and HIV viral load, among which 8 (88%) responded. Four of 16 patients did not receive chemotherapy against medical advice. The chemotherapy regimen included CHOP (n=6), CEOP-B (n=3), m-BACOD (n=2), or CODOX-M/IVAC (n=1). Four patients who refused chemotherapy were lost to follow up. Of 12 patients treated with chemotherapy, 7 were alive in remission, 1 alive in disease, 1 died of treatment related complication, 1 died of progressive lymphoma, 2 died of AIDS related causes. The response to chemotherapy included CR in 7 (70%), PR in 2 (20%) and PD 1 (10%). The median follow-up duration was 15.2 months (range, 0.5–53.2) and the median survival time 43.9 months.