ALBERT

Description: Key Points The genetic cause of SCID impacts on survival and immune reconstitution and should be considered in tailoring HCT for individual patients. Total and naive CD4+ cell counts in SCID patients 6 and 12 months post-HCT predict long-term survival and sustained immune reconstitution.

Description: Abstract 497 Patients with dyskeratosis congenita (DC), a disorder of telomere maintenance, suffer premature degeneration of multiple tissues. Bone marrow failure is the principal cause of mortality, and allogeneic stem cell transplantation is limited by increased treatment-related mortality. Somatic cells can be reprogrammed using defined genetic and chemical factors, yielding “induced pluripotent stem” (iPS) cell lines which have the capacity to differentiate into any tissue. Patient-specific iPS cells therefore hold promise as therapeutic agents and disease models for human degenerative disorders like DC. A cardinal feature of iPS cells is acquisition of indefinite self-renewal capacity, and we have found that telomere length is increased in human iPS cells relative to the normal primary somatic cells from which they are derived. Here we investigated whether defects in telomerase function would limit derivation or self-renewal of iPS cells from patients with DC. We reprogrammed primary fibroblasts from patients with X-linked and autosomal dominant DC, caused by mutations in the genes encoding dyskerin and telomerase RNA component (TERC), respectively. We were able to establish multiple DC-specific iPS lines showing all hallmarks of pluripotency, including the formation of hematopoietic progenitors in vitro. Unexpectedly, DC-specific iPS cells were able to sustain continual proliferation in vitro, in contrast to the premature senescence displayed by the DC fibroblasts. Although early passage DC iPS cells had shorter telomeres than donor fibroblasts, we found that telomere length in DC iPS cells increased with continued passage in culture. To explain this finding, we discovered that steady state levels of TERC, which are critically limiting in several forms of DC, are upregulated in normal and DC iPS cells. We found that TERC upregulation is a feature of the pluripotent state, that the TERC locus is a target of pluripotency-associated transcription factors, and that transcriptional silencing accompanies a 3' deletion at the TERC locus in autosomal dominant DC. Our results demonstrate that reprogramming restores self-renewal capacity in DC cells despite genetic lesions affecting telomerase, and suggest that strategies to enhance endogenous TERC expression may be feasible and therapeutically beneficial in DC patients. The studies demonstrate the value of patient-specific iPS cells for basic and translational discovery, and further the rationale for autologous iPS based cellular therapy of genetic hematologic disorders. Disclosures: Daley: MPM Capital: Consultancy; Solasia: Consultancy; Epizyme: Consultancy; iPierian: Consultancy, Equity Ownership.

Description: Shwachman-Diamond Syndrome (SDS) is an autosomal recessive disorder characterized by pancreatic insufficiency and variable degrees of neutropenia. Additional clinical features include short stature, skeletal abnormalities and bone marrow dysfunction. SDS patients are at increased risk of developing myelodysplasia, aplastic anemia and leukemic transformation. The role and timing of allogeneic hematopoietic stem cell transplantation (HSCT) in SDS remains controversial due to a poorly defined predisposition toward peri-transplant complications and overall poor survival. Here we report three SDS patients (age 13 mo, 16 mo and 8 yr) with severe aplasia successfully transplanted using 5/6 HLA matched unrelated umbilical cord blood. All patients received a previously described “cardiac sparing” conditioning regimen consisting of Melphalan (180 mg/m2), Etoposide (1200 mg/m2), anti-thymocyte globulin (90 mg/kg), and low dose total lymphoid irradiation (500 cGy) with graft versus host disease (GVHD) prophylaxis consisting of cyclosporine and prednisone. Patients received grafts containing 6.7 – 9.1 x 105 CD34+ cells/kg. Myeloid engraftment occurred promptly with the ANC 〉 500 cells/mm3 on day 15 ± 5. Platelet recovery (〉20k without transfusion) occurred on day 20, 30 and 140 days post transplant. All patients displayed 100% donor chimerism by 2 months post transplant. Patients were discharged between 25 – 60 days post transplant without severe complications, though all patients displayed grade II or III acute GVHD, and one developed chronic GVHD. The patients are alive 85, 390 and 850 days post transplant. Factors that may be important in HSCT outcome for SDS include transplantation at a relatively young age prior to malignant transformation, avoidance of cyclophosphamide in the preparative regimen, and adequate GVHD prophylaxis. Importantly, these cases also suggest that unrelated umbilical cord blood, in the absence of a matched family member, should be considered as the preferred source of donor stem cells in SDS patients undergoing HSCT.

Description: Dyskeratosis congenita (DC) is a premature aging syndrome characterized by progressive bone marrow failure, abnormal skin pigmentation and nail dystrophy. We have recently described an autosomal dominant form of DC (AD DC) in a large three-generation kindred that is due to a mutation in the gene encoding human telomerase RNA (hTR). Importantly, we have noted progressive shortening of telomeres in lymphocytes from the most recent generation, correlating with earlier onset of severe cytopenias in some of these patients. While telomere shortening is a normal consequence of the aging process, DC patients display accelerated telomere shortening in many somatic cell types. Allogeneic hematopoietic stem cell transplant (HSCT) remains the only curative therapy for marrow failure in DC. However, HSCT in DC is generally poorly tolerated and associated with significant morbidity, perhaps as a consequence of increased sensitivity of dividing cells to cytotoxic agents. To test this hypothesis, we characterized lymphocytes from nine AD DC patients and age matched controls that had been placed in long term culture following in vitro exposure to irradiation (137Cs) and varying doses of Taxol. Cell proliferation and viability were quantitated by direct visual counting on a hemocytometer, and flow cytometry was employed to assess apoptosis and cell surface expression of senescent markers. CD57 and CD95, markers of cellular senescence and apoptosis, were significantly upregulated on DC T lymphocytes after two weeks in culture relative to controls. In addition to DC lymphocytes having a decreased proliferative capacity, an increased sensitivity to Taxol was noted, with an average decrease of 21% in cell growth relative to similarly treated control cells. This effect was also noted in irradiated DC cells. Finally, DC lymphocytes displayed an increased apoptotic index in the presence of varying doses of Taxol. These results suggest that telomere shortening may be an important factor in determining cellular tolerance to cytotoxic therapy and support the concept of reduced intensity HSCT regimens in both aged individuals and DC patients.

Description: Abstract 2359 Introduction: Dyskeratosis congenita (DC) is an inherited multisystem disorder consisting of premature aging, cancer predisposition, bone marrow failure and the characteristic triad of mucosal leukoplakia, skin dyspigmentation and nail dystrophy. Symptomology associated with DC arises as a consequence of mutations within genes associated with telomeres and telomerase activity manifested by critically shortened telomeres in affected cells. We have previously reported a growth disadvantage and increased intracellular oxidative stress in cultured somatic cells obtained from patients with DC. We hypothesize that telomere maintenance is closely linked to dysregulation in oxidative pathways and consequent DNA damage. Our objective was to discern whether pharmacologic intervention to alleviate oxidative stress imparts a protective effect in DC cells. Methods: T lymphocytes from both DC subjects with hTERC mutations and age-matched controls were cultured and expanded in vitro using CD3/CD28 beads. DNA damage to cells was induced using paclitaxel, etoposide, or ionizing radiation during log-phase of cell growth. Cellular proliferation and apoptosis were monitored by cell counting and flow cytometry (FACS) using Annexin V antibody and propidium iodide. Western blotting was used to measure basal and radiation-induced expression of DNA damage response (DDR) proteins, including total p53 and its activated form (serine 15 phosphorylated; p53S15), p21WAF, and phosphorylated H2AX (gH2AX). Level of oxidative stress was determined by FACS using the cell-permeable fluorogenic probe DCFH and dihydroethedium (DHE) detecting reactive oxygen species (ROS). Anti-oxidants, including vitamin E and N acetyl cysteine (NAC), were used in vitro to modulate levels of oxidative stress in control and radiated cells. Results: Comparison of growth curves demonstrated a significant decrease in proliferation of T cells obtained from DC patients versus control T cells. This growth disadvantage was more pronounced following cell exposure to radiation, paclitaxel, and etoposide. To explain these differences we investigated several parameters indicative of DNA damage. DC lymphocytes had higher basal levels of apoptosis, while radiation resulted in comparable levels of apoptosis in both DC and control cultures. Similarly, DDR markers p53 and p53S15, but not p21 and g-H2AX, were basally expressed at higher levels in DC lymphocytes while radiation, in a dose-dependent manner, upregulated expression of p53, p53S15, p21 and g-H2AX in both DC and control lymphocytes. Consistent with DDR data, elevated basal levels of ROS were found in short term DC cultures. Additionally, in a dose dependent manner, the anti-oxidant NAC partially ameliorated the growth disadvantage of DC cells. Importantly, NAC also decreased radiation-induced apoptosis and oxidative stress in DC cells. Studies are ongoing to characterize the modulation of DDR markers in NAC-treated cells. Conclusions: DC is an important disease model for studying the effects of telomere shortening on cellular proliferation and other molecular pathways involved in cell senescence and aging. Our findings of elevated basal levels of apoptosis, DDR proteins and oxidative stress in DC lymphocytes, as well as increased sensitivity of DC cells to cytotoxic agents suggests a role of telomerase and/or telomere length in regulating oxidative and DNA damage response pathways. This data also validates the clinical finding of DC patients' intolerance to myeloablative therapy. Finally a pharmacologic approach to reduce oxidative stress may alleviate some of the untoward toxicities associated with current cytotoxic treatments in DC. Clinical trials testing various anti-oxidant therapies are currently under design. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction Diamond Blackfan Anemia (DBA) is a congenital bone marrow failure disorder characterized by severe erythroblastopenia. Nearly 70% of patients harbor mutations in ribosomal genes that lead to deficiency in ribosomal protein synthesis and impaired red blood cell production. Primary treatment modalities for DBA are steroids or red blood cell transfusions, while allogeneic hematopoietic stem cell (HSC) transplant remains the only curative option. While gene therapy is currently being utilized in a limited number of diseases, the advent of induced pluripotent stem cell (iPSC) technology and the capacity to manufacture HSCs from one’s own fibroblasts could expand the utility of gene correction strategies in disorders of hematopoiesis. We hypothesized that creation of iPSCs from a DBA patient’s fibroblasts could provide an in vitro model of a disease with defective erythroid development, and that correction of the mutation might alleviate the presumed block in erythropoiesis. Methods and Results Creation of iPSCs from DBA patients has been problematic for investigators due to reasons that are not entirely clear. After multiple attempts, five iPS cell lines (using an integration-free, novel 6-factor episomal construct) were created from a DBA patient carrying a non-sense mutation (R94X) in the third exon of RPS19. Experiments characterizing iPSCs ability to generate HSC (via OP9 mouse stromal cell co-culture) and undergo erythropoiesis demonstrated an approximate decrease of ~8-9x production of both CD71+/CD235+ and CD71-/CD235+in DBA cells compared to controls. Multiple strategies have been developed to target the RPS19 mutation of interest and replace it with corrected sequence. The first strategy relies on large targeting vectors that carry 2-4kb of homologous DNA flanking the mutation of interest. These vectors are engineered to carry drug selectable cassettes within the adjacent intron to permit retrieval of cells that have undergone homologous recombination. The second strategy targets the mutation using CRISPR technology using three key components: an endonuclease (Cas9), a chimeric RNA (crRNA + tracrRNA) capable of recognizing a specific genomic sequence and a correction oligo. Additional novel strategies are also underway that provide an avenue towards increased targeting efficiency. All correction strategies were employed in skin fibroblasts and iPSCs from a DBA patient. CRISPR vectors tested in 293T HEK cells and DBA skin fibroblasts have yielded promising results. Using a combination of the SURVEYOR assay and DNA sequencing to test targeting/cutting efficiency, we found the CRISPR vector cuts the mutated allele (designed to target only the mutated allele) in 13% of alleles sequenced. In comparison, vectors designed to cut either allele (targeting does not incorporate mutation of interest) were able to cut 17% of alleles sequenced, suggesting corrected cells may be retrieved without drug selection and minimal intervention. CRISPR correction has now been performed in DBA iPSCs and early experiments have indicated successful targeting in 3.5% of cells. Conclusions CD34+ and erythroid progenitor cells generated from DBA iPSC’s recapitulate disease characteristics and offer a potential source of cells to study disease characteristics in vitro. CRISPR methodology allowed for correction of this specific mutation and studies are currently underway to determine if the correction has rescued the erythopoietic defect. Futhermore, whole genome sequencing studies are needed to determine the background mutagenicity and safety of this approach. This technology may allow for the correction of specific DBA mutations that result in the generation of autologous ‘gene corrected’ hematopoietic stem cells that are suitable for transplantation. Disclosures Townes: University of Alabama at Birmingham: Employment.

Description: Introduction: Dyskeratosis congenita (DC) is an inherited multisystem disorder characterized by the classical clinical triad of leukoplakia, skin dyspigmentation and nail dystrophy with concomitant premature aging, cancer predisposition, and bone marrow failure. Conventional treatments for marrow failure, including transplantation, have been poorly tolerated for DC patients. Several mutations within telomeres/telomerase-associated genes have been attributed to DC, resulting in shortened telomeres. Growing evidence suggests that shortened telomeres are linked to DNA damage responses and subsequent elevation of reactive oxygen species (ROS). We have previously reported increased intracellular ROS and induction of DDR protein expression in TERC-deficient cells. This study was undertaken to confirm these findings in other DC genotypes (TERT, TINF2, DKC1) and provide additional evidence supporting a relationship among telomere shortening, DDR and dysregulation in oxidative pathways. Methods: Somatic cells from DC subjects with TERC (5 patients), TERT (4), DKC1 (1) and TINF2 (1) mutations and age-matched controls were used in these studies. Cell types included lymphocytes and fibroblasts from skin and lung. DC genotypes were confirmed by DNA sequencing, and all DC cells displayed shortened telomeres. T lymphocytes were expanded in vitro using CD3/CD28 beads. Ionizing radiation was used to induce DNA damage. Cell proliferation was assessed on a Nexelom automatic counter. Western blotting was used to measure basal and radiation-induced expression of DNA damage response (DDR) proteins, including total p53 and its activated form (serine 15 phosphorylated; p53S15), ATM, BRCA2 and phosphorylated H2AX (gH2AX). Levels of oxidative stress were determined by FACS using the cell-permeable fluorogenic probe DCFH and dihydroethedium (DHE) detecting reactive oxygen species (ROS). Antioxidant culture conditions, such as N acetyl cysteine (NAC) or low oxygen, were used in vitro to modulate levels of oxidative stress in control and radiated cells. Results: We investigated ROS levels and several parameters indicative of DNA damage in DC lymphocytes and fibroblasts with mutations in TERC, TERT, DKC1 or TINF2. In cells from all DC genotypes a consistent increase in p53 expression and ROS levels was noted. Basal expression of p53 was increased by at least 50% in DC lymphocytes compared to controls. Additionally, basal ROS levels from TERC and TINF2 mutated lymphocytes were significantly increased compared to controls. While the degree of radiation-induced DDR protein expression was similar in DC compared to control cells, ROS remained significantly higher in DC-irradiated lymphocytes. Lung fibroblasts from a DKC1 patient demonstrated increased ROS and reduced proliferative capacity compared to controls. Importantly, NAC decreased DDR in DC lymphocytes while a low oxygen environment markedly improved the proliferative capacity of both DC lymphocytes and lung fibroblasts. Conclusions: DC is an important human disease model for studying the effects of telomere shortening on pathways involved in cell senescence and aging. Our findings indicate elevated basal and irradiation-induced levels of p53 expression and ROS in DC cells regardless of the underlying mutation. This suggests a role for telomerase deficiency and/or shortened telomeres in regulating oxidative and DNA damage response pathways. Furthermore, reducing ROS may serve as an adjunctive therapeutic approach to limit toxicity of bone marrow transplant regimens. Clinical trials are needed to evaluate the impact of antioxidant therapies in this disease. Disclosures No relevant conflicts of interest to declare.

Description: Hydroxychloroquine (HCQ), a lysosomotropic amine, is an immunosuppressive agent presently being evaluated in bone marrow transplant patients to treat graft-versus-host disease. While its immunosuppressive properties have been attributed primarily to its ability to interfere with antigen processing, recent reports demonstrate HCQ also blocks T-cell activation in vitro. To more precisely define the T-cell inhibitory effects of HCQ, the authors evaluated T-cell antigen receptor (TCR) signaling events in a T-cell line pretreated with HCQ. In a concentration-dependent manner, HCQ inhibited anti-TCR–induced up-regulation of CD69 expression, a distal TCR signaling event. Proximal TCR signals, including inductive protein tyrosine phosphorylation, tyrosine phosphorylation of phospholipase C γ1, and total inositol phosphate production, were unaffected by HCQ. Strikingly, anti-TCR-crosslinking–induced calcium mobilization was significantly inhibited by HCQ, particularly at the highest concentrations tested (100 μmol/L) in both T-cell lines and primary T cells. HCQ, in a dose-dependent fashion, also reduced a B-cell antigen receptor calcium signal, indicating this effect may be a general property of HCQ. Inhibition of the calcium signal correlated directly with a reduction in the size of thapsigargin-sensitive intracellular calcium stores in HCQ-treated cells. Together, these findings suggest that disruption of TCR-crosslinking–dependent calcium signaling provides an additional mechanism to explain the immunomodulatory properties of HCQ.

Description: Wiskott-Aldrich syndrome (WAS) is an X-linked disease caused by mutations in the WAS gene, leading to thrombocytopenia, eczema, recurrent infections, autoimmune disease, and malignancy. Hematopoietic cell transplantation (HCT) is the primary curative approach, with the goal of correcting the underlying immunodeficiency and thrombocytopenia. HCT outcomes have improved over time, particularly for patients with HLA-matched sibling and unrelated donors. We report the outcomes of 129 patients with WAS who underwent HCT at 29 Primary Immune Deficiency Treatment Consortium centers from 2005 through 2015. Median age at HCT was 1.2 years. Most patients (65%) received myeloablative busulfan-based conditioning. With a median follow-up of 4.5 years, the 5-year overall survival (OS) was 91%. Superior 5-year OS was observed in patients

Description: Dyskeratosis congenita (DC) is an inherited bone marrow (BM) failure syndrome associated with mutations in telomerase genes and the acquisition of shortened telomeres in blood cells. To investigate the basis of the compromised hematopoiesis seen in DC, we analyzed cells from granulocyte colony-stimulating factor mobilized peripheral blood (mPB) collections from 5 members of a family with autosomal dominant DC with a hTERC mutation. Premobilization BM samples were hypocellular, and percentages of CD34+ cells in marrow and mPB collections were significantly below values for age-matched controls in 4 DC subjects. Directly clonogenic cells, although present at normal frequencies within the CD34+ subset, were therefore absolutely decreased. In contrast, even the frequency of long-term culture-initiating cells within the CD34+ DC mPB cells was decreased, and the telomere lengths of these cells were also markedly reduced. Nevertheless, the different lineages of mature cells were produced in normal numbers in vitro. These results suggest that marrow failure in DC is caused by a reduction in the ability of hematopoietic stem cells to sustain their numbers due to telomere impairment rather than a qualitative defect in their commitment to specific lineages or in the ability of their lineage-restricted progeny to execute normal differentiation programs.