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Inherited bone marrow failure syndromes (IBMFSs) are a group of disorders typified by impaired production of 1 or several blood cell types. The telomere biology disorders dyskeratosis congenita (DC) and its severe variant, Høyeraal-Hreidarsson (HH) syndrome, are rare IBMFSs characterized by bone marrow failure, developmental defects, and various premature aging complications associated with critically short telomeres. We identified biallelic variants in the gene encoding the 5′-to-3′ DNA exonuclease Apollo/SNM1B in 3 unrelated patients presenting with a DC/HH phenotype consisting of early-onset hypocellular bone marrow failure, B and NK lymphopenia, developmental anomalies, microcephaly, and/or intrauterine growth retardation. All 3 patients carry a homozygous or compound heterozygous (in combination with a null allele) missense variant affecting the same residue L142 (L142F or L142S) located in the catalytic domain of Apollo. Apollo-deficient cells from patients exhibited spontaneous chromosome instability and impaired DNA repair that was complemented by CRISPR/Cas9-mediated gene correction. Furthermore, patients’ cells showed signs of telomere fragility that were not associated with global reduction of telomere length. Unlike patients’ cells, human Apollo KO HT1080 cell lines showed strong telomere dysfunction accompanied by excessive telomere shortening, suggesting that the L142S and L142F Apollo variants are hypomorphic. Collectively, these findings define human Apollo as a genome caretaker and identify biallelic Apollo variants as a genetic cause of a hitherto unrecognized severe IBMFS that combines clinical hallmarks of DC/HH with normal telomere length.

Inherited bone marrow failure syndromes (IBMFSs) are a group of disorders typified by impaired production of 1 or several blood cell types. The telomere biology disorders dyskeratosis congenita (DC) and its severe variant, Høyeraal-Hreidarsson (HH) syndrome, are rare IBMFSs characterized by bone marrow failure, developmental defects, and various premature aging complications associated with critically short telomeres. We identified biallelic variants in the gene encoding the 5′-to-3′ DNA exonuclease Apollo/SNM1B in 3 unrelated patients presenting with a DC/HH phenotype consisting of early-onset hypocellular bone marrow failure, B and NK lymphopenia, developmental anomalies, microcephaly, and/or intrauterine growth retardation. All 3 patients carry a homozygous or compound heterozygous (in combination with a null allele) missense variant affecting the same residue L142 (L142F or L142S) located in the catalytic domain of Apollo. Apollo-deficient cells from patients exhibited spontaneous chromosome instability and impaired DNA repair that was complemented by CRISPR/Cas9-mediated gene correction. Furthermore, patients' cells showed signs of telomere fragility that were not associated with global reduction of telomere length. Unlike patients' cells, human Apollo KO HT1080 cell lines showed strong telomere dysfunction accompanied by excessive telomere shortening, suggesting that the L142S and L142F Apollo variants are hypomorphic. Collectively, these findings define human Apollo as a genome caretaker and identify biallelic Apollovariants as a genetic cause of a hitherto unrecognized severe IBMFS that combines clinical hallmarks of DC/HH with normal telomere length.

The leukemic population in 63 patients with acute myeloid leukemia (AML) was studied with 15 monoclonal antibodies that detect lineage-related and stage-related antigens on normal hemopoietic cells. Indirect immunofluorescence and fluorescence-activated cell sorting showed that subpopulations of leukemic cells reacted with some or all antibodies, but the percentage of cells reacting with a single antibody varied widely among patients. The composite antigenic phenotype of the various cases, as determined by immunofluorescence assay, did not correlate with the French-American-British morphological classification. Furthermore, some cells in each case failed to express any antigen normally expressed on myelomonocytic precursors from the level of the early CFU-GM to the mature granulocyte or monocyte. In double-fluorescence experiments, the individual cells expressed none, one, or both antigens. These results demonstrate that there is considerable subpopulation heterogeneity in AML. This heterogeneity may considerably limit or complicate the use of monoclonal antibodies for diagnosis, prognosis, and treatment of acute nonlymphocytic leukemia (ANLL).

The leukemic population in 63 patients with acute myeloid leukemia (AML) was studied with 15 monoclonal antibodies that detect lineage- related and stage-related antigens on normal hemopoietic cells. Indirect immunofluorescence and fluorescence-activated cell sorting showed that subpopulations of leukemic cells reacted with some or all antibodies, but the percentage of cells reacting with a single antibody varied widely among patients. The composite antigenic phenotype of the various cases, as determined by immunofluorescence assay, did not correlate with the French-American-British morphological classification. Furthermore, some cells in each case failed to express any antigen normally expressed on myelomonocytic precursors from the level of the early CFU-GM to the mature granulocyte or monocyte. In double-fluorescence experiments, the individual cells expressed none, one, or both antigens. These results demonstrate that there is considerable subpopulation heterogeneity in AML. This heterogeneity may considerably limit or complicate the use of monoclonal antibodies for diagnosis, prognosis, and treatment of acute nonlymphocytic leukemia (ANLL).

A monoclonal antibody, L5.1, obtained by immunizing a Balb/c mouse with HL60 human promyelocytic leukemia cells, was found to react with both HL60 cells and with the K562(S) cell line. This monoclonal antibody binds and immunoprecipitates a glycoprotein (Mr 87,000) present on the cell surface membrane of K562(S) as a disulfide bonded dimer. In competition experiments L5.1 competes with both transferrin and OKT9 (a known antitransferrin receptor antibody) for binding to target K562(S) erythroleukemia cells. Binding of both L5.1 and transferrin to the surface of K562(S) cells is inhibited by treatment with 12--O- tetradecanoyl-phorbol-13-acetate, and the extent and time course of inhibition is similar in both cases. Cell sorting analysis of normal human marrow cells incubated with L5.1 indicates that L5.1 reacts strongly with all the morphologically recognizable erythroid lineage precursors, from the pronormoblast to the orthochromatic normoblast, and with reticulocytes. Erythrocytes, myeloid elements, monocytes, megakaryocytes and platelets, peripheral blood B and T lymphocytes do not bind significantly with this antibody and only a small fraction of promyelocytes was reactive. Antibody L5.1 did not react with leukemic cells of patients with acute lymphoblastic, myeloblastic and promyelocytic leukemias, but it did react with some established B (1 of 5) and T (2 of 3) cell lines, and a myeloid (1 of 3) cell line, and with PHA-stimulated peripheral blood lymphocytes. The nonhemopoietic cell lines tested did not bind with L5.1 with the exception of a colorectal adenocarcinoma and a melanoma cell line, which were both strongly positive. The relationship of antibody L5.1 to other monoclonal antibodies that bind the transferrin receptor is discussed.

A monoclonal antibody, L5.1, obtained by immunizing a Balb/c mouse with HL60 human promyelocytic leukemia cells, was found to react with both HL60 cells and with the K562(S) cell line. This monoclonal antibody binds and immunoprecipitates a glycoprotein (Mr 87,000) present on the cell surface membrane of K562(S) as a disulfide bonded dimer. In competition experiments L5.1 competes with both transferrin and OKT9 (a known antitransferrin receptor antibody) for binding to target K562(S) erythroleukemia cells. Binding of both L5.1 and transferrin to the surface of K562(S) cells is inhibited by treatment with 12–O–tetradecanoyl-phorbol–13–acetate, and the extent and time course of inhibition is similar in both cases. Cell sorting analysis of normal human marrow cells incubated with L5.1 indicates that L5.1 reacts strongly with all the morphologically recognizable erythroid lineage precursors, from the pronormoblast to the orthochromatic normoblast, and with reticulocytes. Erythrocytes, myeloid elements, monocytes, megakaryocytes and platelets, peripheral blood B and T lymphocytes do not bind significantly with this antibody and only a small fraction of promyelocytes was reactive. Antibody L5.1 did not react with leukemic cells of patients with acute lymphoblastic, myeloblastic and promyelocytic leukemias, but it did react with some established B (1 of 5) and T (2 of 3) cell lines, and a myeloid (1 of 3) cell line, and with PHA-stimulated peripheral blood lymphocytes. The nonhemopoietic cell lines tested did not bind with L5.1 with the exception of a colorectal adenocarcinoma and a melanoma cell line, which were both strongly positive. The relationship of antibody L5.1 to other monoclonal antibodies that bind the transferrin receptor is discussed.

The ability for siRNA containing lentiviral vectors to specifically downregulate gene expression in the hematopoietic system following hematopoietic stem cell transplantation provides a new tool for developing disease models of leukemia and other hematological disorders. The tumor suppressor gene, p53, has been implicated in the genesis or progression of hematological malignancies, such as AML, MDS, and CML, and disorders such as myeloproliferative disease (MPD). Following transduction of Ly5.1 hematopoietic stem cells using a lentiviral vector expressing small interfering RNAs specific for mouse p53, cells were transplanted into lethally irradiated syngeneic recipient mice. Peripheral blood analyses by FACS at 7 weeks posttransplant showed significantly increased numbers of Gr1 Mac1 double positive progenitor like cells in 4 of 4 mice (73±14% compared to control siGFP transplanted mice 5±0.5%). Interestingly, 3 of 4 transplanted mice showed signs of morbidity by 16 weeks posttransplant due to a myelogenous proliferative disease (MPD) phenotype characterized by high peripheral blood neutrophil counts (>70% of white blood cells) and anemia (<106 RBC/μL), and extensive neutrophil infiltration into spleen and liver. Furthermore, peripheral blood smears show an expansion of megakaryocytes. Bone marrow mononuclear cells from transplanted mice were analyzed for p53 protein expression following p53 stabilization by irradiation and significantly lower levels of p53 were detected in sip53-transduced mice compared to siGFP lentiviral vector control mice. No altering of normal hematopoietic cell differentiation was observed in control mice from nonspecific RNAi interactions, as determined by FACS or blood cell count analyses. These results demonstrate that downregulated expression of p53 in mice hematopoietic cells yields a myelogenous proliferative disease and creates a model for the study of p53 involvement in the genesis and progression of hematological disorders. Furthermore, these results demonstrate the ability of lentiviral vector mediated siRNA to efficiently downregulate endogenous gene expression in vivo and model human disease.

The ability for siRNA containing lentiviral vectors to specifically downregulate gene expression in the hematopoietic system following hematopoietic stem cell transplantation provides a new tool for developing disease models of leukemia and other hematological disorders. The tumor suppressor gene, p53, has been implicated in the genesis or progression of hematological malignancies, such as AML, MDS, and CML, and disorders such as myeloproliferative disease (MPD). Following transduction of Ly5.1 hematopoietic stem cells using a lentiviral vector expressing small interfering RNAs specific for mouse p53, cells were transplanted into lethally irradiated syngeneic recipient mice. Peripheral blood analyses by FACS at 7 weeks posttransplant showed significantly increased numbers of Gr1 Mac1 double positive progenitor like cells in 4 of 4 mice (73±14% compared to control siGFP transplanted mice 5±0.5%). Interestingly, 3 of 4 transplanted mice showed signs of morbidity by 16 weeks posttransplant due to a myelogenous proliferative disease (MPD) phenotype characterized by high peripheral blood neutrophil counts (>70% of white blood cells) and anemia (<106RBC/μL), and extensive neutrophil infiltration into spleen and liver. Furthermore, peripheral blood smears show an expansion of megakaryocytes. Bone marrow mononuclear cells from transplanted mice were analyzed for p53 protein expression following p53 stabilization by irradiation and significantly lower levels of p53 were detected in sip53-transduced mice compared to siGFP lentiviral vector control mice. No altering of normal hematopoietic cell differentiation was observed in control mice from nonspecific RNAi interactions, as determined by FACS or blood cell count analyses. These results demonstrate that downregulated expression of p53 in mice hematopoietic cells yields a myelogenous proliferative disease and creates a model for the study of p53 involvement in the genesis and progression of hematological disorders. Furthermore, these results demonstrate the ability of lentiviral vector mediated siRNA to efficiently downregulate endogenous gene expression in vivo and model human disease.