Hisham Abdel-Azim

β-globin gene transfer to human bone marrow for sickle cell disease

Autologous hematopoietic stem cell gene therapy is an approach to treating sickle cell disease (SCD) patients that may result in lower morbidity than allogeneic transplantation. We examined the potential of a lentiviral vector (LV) (CCL-βAS3-FB) encoding a human hemoglobin (HBB) gene engineered to impede sickle hemoglobin polymerization (HBBAS3) to transduce human BM CD34+ cells from SCD donors and prevent sickling of red blood cells produced by in vitro differentiation. The CCL-βAS3-FB LV transduced BM CD34+ cells from either healthy or SCD donors at similar levels, based on quantitative PCR and colony-forming unit progenitor analysis. Consistent expression of HBBAS3 mRNA and HbAS3 protein compromised a fourth of the total β-globin-like transcripts and hemoglobin (Hb) tetramers. Upon deoxygenation, a lower percentage of HBBAS3-transduced red blood cells exhibited sickling compared with mock-transduced cells from sickle donors. Transduced BM CD34+ cells were transplanted into immunodeficient mice, and the human cells recovered after 2-3 months were cultured for erythroid differentiation, which showed levels of HBBAS3 mRNA similar to those seen in the CD34+ cells that were directly differentiated in vitro. These results demonstrate that the CCL-βAS3-FB LV is capable of efficient transfer and consistent expression of an effective anti-sickling β-globin gene in human SCD BM CD34+ progenitor cells, improving physiologic parameters of the resulting red blood cells.

Obesity is associated with residual leukemia following induction therapy for childhood B- precursor acute lymphoblastic leukemia

Obesity is associated with poorer event-free survival (EFS) in pediatric acute lymphoblastic leukemia (ALL). Persistent minimal residual disease (MRD) in the bone marrow as measured by multidimensional flow cytometry (MDF) is a key early prognostic indicator and is strongly associated with EFS. We therefore hypothesized that obesity during induction would be associated with positive end-of-induction MRD (≥0.01%). We analyzed MDF of end-induction bone marrow samples from a historical cohort of 198 children newly diagnosed with B-precursor ALL (BP-ALL) and treated with Childrens Oncology Group induction regimens. We assessed the influence of body mass index on risk for positive end-induction MRD in the bone marrow. In our cohort of BP-ALL, 30 children (15.2%) were overweight and 41 (20.7%) were obese at diagnosis. Independent of established predictors of treatment response, obesity during induction was associated with significantly greater risk for persistent MRD (odds ratio, 2.57; 95% confidence interval, 1.19 to 5.54; P = .016). Obesity and overweight were associated with poorer EFS irrespective of end-induction MRD (P = .012). Obese children with newly diagnosed BP-ALL are at increased risk for positive end-induction MRD and poorer EFS.

Transplantation for children with acute myeloid leukemia: a comparison of outcomes with reduced intensity and myeloablative regimens

The safety and efficacy of reduced-intensity conditioning (RIC) regimens for the treatment of pediatric acute myeloid leukemia is unknown. We compared the outcome of allogeneic hematopoietic cell transplantation in children with acute myeloid leukemia using RIC regimens with those receiving myeloablative-conditioning (MAC) regimens. A total of 180 patients were evaluated (39 with RIC and 141 with MAC regimens). Results of univariate and multivariate analysis showed no significant differences in the rates of acute and chronic graft-versus-host disease, leukemia-free, and overall survival between treatment groups. The 5-year probabilities of overall survival with RIC and MAC regimens were 45% and 48%, respectively (P = .99). Moreover, relapse rates were not higher with RIC compared with MAC regimens (39% vs 39%; P = .95), and recipients of MAC regimens were not at higher risk for transplant-related mortality compared with recipients of RIC regimens (16% vs 16%; P = .73). After carefully controlled analyses, we found that in this relatively modest study population, the data supported a role for RIC regimens for acute myeloid leukemia in children undergoing allogeneic hematopoietic cell transplantation. The data also provided justification for designing a carefully controlled randomized clinical trial that examines the efficacy of regimen intensity in this population.

Clinical and genetic heterogeneity in Omenn syndrome and severe combined immune deficiency.

Abstract:  OS has been described as a clinical phenotype in infants characterized by SCID, diffuse erythroderma, and other distinct features. The pathogenesis is secondary to autologous, auto‐reactive T cells produced as rare escapees from the SCID blockade. Mutations in either the RAG1 or RAG2 gene that lead to partial recombinase activity are responsible for many of the patients with these clinical features. We report on two patients, one with an atypical phenotype of OS (absence of rash but presence of other typical features) who harbored a previously undescribed mutation in RAG1, and a second who had many of the classic features of OS but was found to have a mutation in the common gamma chain (γc) cytokine receptor gene. These cases highlight the clinical and genetic heterogeneity of OS.

A prognostic model predicting autologous transplantation outcomes in children, adolescents and young adults with Hodgkin lymphoma

Autologous hematopoietic cell transplantation (AutoHCT) is a potentially curative treatment modality for relapsed/refractory Hodgkin lymphoma (HL). However, no large studies have evaluated pretransplant factors predictive of outcomes of AutoHCT in children, adolescents and young adults (CAYA, age <30 years). In a retrospective study, we analyzed 606 CAYA patients (median age 23 years) with relapsed/refractory HL who underwent AutoHCT between 1995 and 2010. The probabilities of PFS at 1, 5 and 10 years were 66% (95% confidence interval (CI): 62–70), 52% (95% CI: 48–57) and 47% (95% CI: 42–51), respectively. Multivariate analysis for PFS demonstrated that at the time of AutoHCT patients with Karnofsky/Lansky score ⩾90, no extranodal involvement and chemosensitive disease had significantly improved PFS. Patients with time from diagnosis to first relapse of <1 year had a significantly inferior PFS. A prognostic model for PFS was developed that stratified patients into low-, intermediate- and high-risk groups, predicting for 5-year PFS probabilities of 72% (95% CI: 64–80), 53% (95% CI: 47–59) and 23% (95% CI: 9–36), respectively. This large study identifies a group of CAYA patients with relapsed/refractory HL who are at high risk of progression after AutoHCT. Such patients should be targeted for novel therapeutic and/or maintenance approaches post-AutoHCT.

The incidence, mortality and timing of Pneumocystis jiroveci pneumonia after hematopoietic cell transplantation: a CIBMTR analysis.

Pneumocystis jiroveci pneumonia (PJP) is associated with high morbidity and mortality after hematopoietic stem cell transplantation (HSCT). Little is known about PJP infections after HSCT because of the rarity of disease given routine prophylaxis. We report the results of a Center for International Blood and Marrow Transplant Research study evaluating the incidence, timing, prophylaxis agents, risk factors and mortality of PJP after autologous (auto) and allogeneic (allo) HSCT. Between 1995 and 2005, 0.63% allo recipients and 0.28% auto recipients of first HSCT developed PJP. Cases occurred as early as 30 days to beyond a year after allo HSCT. A nested case cohort analysis with supplemental data (n=68 allo cases, n=111 allo controls) revealed that risk factors for PJP infection included lymphopenia and mismatch after HSCT. After allo or auto HSCT, overall survival was significantly poorer among cases vs controls (P=0.0004). After controlling for significant variables, the proportional hazards model revealed that PJP cases were 6.87 times more likely to die vs matched controls (P<0.0001). We conclude PJP infection is rare after HSCT but is associated with high mortality. Factors associated with GVHD and with poor immune reconstitution are among the risk factors for PJP and suggest that protracted prophylaxis for PJP in high-risk HSCT recipients may improve outcomes.

Transcriptional Regulation of JARID1B/KDM5B Histone Demethylase by Ikaros, Histone Deacetylase 1 (HDAC1), and Casein Kinase 2 (CK2) in B-cell Acute Lymphoblastic Leukemia

Impaired function of the Ikaros (IKZF1) protein is associated with the development of high-risk B-cell precursor acute lymphoblastic leukemia (B-ALL). The mechanisms of Ikaros tumor suppressor activity in leukemia are unknown. Ikaros binds to the upstream regulatory elements of its target genes and regulates their transcription via chromatin remodeling. Here, we report that Ikaros represses transcription of the histone H3K4 demethylase, JARID1B (KDM5B). Transcriptional repression of JARID1B is associated with increased global levels of H3K4 trimethylation. Ikaros-mediated repression of JARID1B is dependent on the activity of the histone deacetylase, HDAC1, which binds to the upstream regulatory element of JARID1B in complex with Ikaros. In leukemia, JARID1B is overexpressed, and its inhibition results in cellular growth arrest. Ikaros-mediated repression of JARID1B in leukemia is impaired by pro-oncogenic casein kinase 2 (CK2). Inhibition of CK2 results in increased binding of the Ikaros-HDAC1 complex to the promoter of JARID1B, with increased formation of trimethylated histone H3 lysine 27 and decreased histone H3 Lys-9 acetylation. In cases of high-risk B-ALL that carry deletion of one Ikaros (IKZF1) allele, targeted inhibition of CK2 restores Ikaros binding to the JARID1B promoter and repression of JARID1B. In summary, the presented data suggest a mechanism through which Ikaros and HDAC1 regulate the epigenetic signature in leukemia: via regulation of JARID1B transcription. The presented data identify JARID1B as a novel therapeutic target in B-ALL and provide a rationale for the use of CK2 inhibitors in the treatment of high-risk B-ALL.

Infection Rates among Acute Leukemia Patients Receiving Alternative Donor Hematopoietic Cell Transplantation.

Alternative graft sources (umbilical cord blood [UCB], matched unrelated donors [MUD], or mismatched unrelated donors [MMUD]) enable patients without a matched sibling donor to receive potentially curative hematopoietic cell transplantation (HCT). Retrospective studies demonstrate comparable outcomes among different graft sources. However, the risk and types of infections have not been compared among graft sources. Such information may influence the choice of a particular graft source. We compared the incidence of bacterial, viral, and fungal infections in 1781 adults with acute leukemia who received alternative donor HCT (UCB, n= 568; MUD, n = 930; MMUD, n = 283) between 2008 and 2011. The incidences of bacterial infection at 1 year were 72%, 59%, and 65% (P < .0001) for UCB, MUD, and MMUD, respectively. Incidences of viral infection at 1 year were 68%, 45%, and 53% (P < .0001) for UCB, MUD, and MMUD, respectively. In multivariable analysis, bacterial, fungal, and viral infections were more common after either UCB or MMUD than after MUD (P < .0001). Bacterial and viral but not fungal infections were more common after UCB than MMUD (P = .0009 and <.0001, respectively). The presence of viral infection was not associated with an increased mortality. Overall survival (OS) was comparable among UCB and MMUD patients with Karnofsky performance status (KPS) ≥ 90% but was inferior for UCB for patients with KPS < 90%. Bacterial and fungal infections were associated with poorer OS. Future strategies focusing on infection prevention and treatment are indicated to improve HCT outcomes.

Galectin-3 in pre-B acute lymphoblastic leukemia

Only 30–40% of adult patients with acute lymphoblastic leukemia (ALL) achieve long-term, disease-free survival and can be considered cured. The remainder will eventually relapse and die from disease progression or other ALL-related consequences.1,2 Relapse most frequently originates in the bone marrow (BM),3 where stroma protects ALL cells against drug treatment.4 The development of drug resistance in ALL cells as occurs in vivo can be modeled by coculture of ALL cells in the presence of irradiated stromal cells during drug exposure. We have shown previously that this allows for the outgrowth of drug-insensitive leukemic cells (e.g. Zhang et al5), in a process that has been named environmental-mediated drug resistance. Microarray analysis on precursor B-lineage (pre-B) ALL cells from a BCR/ABL transgenic mouse model for Ph-positive ALL undergoing environmental-mediated drug resistance showed significantly increased expression of mRNAs related to stress and inflammation.6 Among others, we found that transcript levels of the Galectin-3 gene (Lgals3) were significantly increased upon development of resistance to the Bcr/Abl-targeted tyrosine kinase inhibitor nilotinib, and this was also seen in pre-B ALL cells isolated from the BM of BCR/ABL transgenic mice that had been treated with nilotinib for 8 days.6 Galectin-3, a 30-kDa protein without enzymatic activity, belongs to the family of lectins, which recognize and specifically bind (mostly extracellular) carbohydrate structures. Galectin-3 has a C-terminal carbohydrate-binding domain with preference for galactose and poly-N-acetyllactosamine-modified glycans. Its N-terminal domain promotes homotypic oligomerization. Because of these domains, extracellular Galectin-3 can promote the cross-linking and lattice formation between glycan-containing molecules on the cell surface.7 ALL cells were reported not to express Galectin-3.8 The induction of its transcripts in the pre-B ALL cells prompted us to further examine this. We first assessed expression of Galectin-3 protein in BM samples from ALL patients using fluorescence-activated cell sorting (FACS). Galectin-3 was clearly present in pre-B cells from BM of all four ALL patients (Figure 1a). Elevated serum levels of Galectin-3 were reported in some types of cancer such as metastatic colorectal cancer and melanoma.9,10 To evaluate circulating levels of Galectin-3 in ALL patients, plasma samples from peripheral blood and BM were collected from ALL patients, as well as controls. Interestingly, we found that plasma levels of Galectin-3 in BM from ALL patients were significantly higher than healthy controls (P<0.05) (Figure 1b) as detected by enzyme-linked immunosorbent assay. To further investigate circumstances under which Galectin-3 levels are increased, we used human patient-derived pre-B ALL cells that were passaged in NSG mice and grown ex vivo in the presence of OP9 stromal feeder cells. In this system, the ALL cells adhere to the stromal cells, migrate underneath them and return into the medium in a dynamic fashion. Notably, cells that were harvested from underneath the OP9 layer, and had thus been in direct contact with OP9 cells, expressed a high amount of Galectin-3, while cells in suspension or cells cultured without OP9 for 24 h had low expression of cell surface or intracellular Galectin-3 as measured by FACS (Figure 1c). OP9 cells secrete Galectin-3 (Supplementary Figure 1) and part of the Galectin-3 detected on these ALL cells was of stromal origin (Fei F, Lim M, Groffen J, Heisterkamp N, manuscript in preparation). Figure 1 Expression of Galectin-3 in pre-B ALL samples. (a) Total (cell surface + intracellular) expression of Galectin-3 in CD19+ CD10+ double-positive cells from primary ALL patient BM samples as assessed by FACS in fixed permeabilized cells. Control, isotype-matched ... As increased cellular levels of Galectin-3 are protective to several cancer cell types,8,11,12 we elevated Galectin-3 levels in ALL cells by transduction of TXL2 cells with pMIG-Gal3 and control vector pMIG. As phosphorylation of serine residue 6 in Galectin-3 is needed for this protein to provide protection to BT-549 breast cancer cells against cisplatin-induced apoptosis,12 we also compared the effect of a Gal3S6A mutant. Analysis of major signal transduction pathways in TXL2-pMIG, TXL2-pMIG-Gal3 and TXL2-pMIG-Gal3S6A cocultured with or without OP9 cells did not show major differences between these cells and their growth was also not statistically significantly different (Supplementary Figure 2). Interestingly, compared with TXL2-pMIG and TXL2-pMIG-Gal3 cells, TXL2-pMIG-Gal3-S6A cells were more vulnerable to spontaneous apoptosis when cultured without OP9 cells over a 3-day period (Figure 2a). Consistent with studies in other hematopoietic cell type-derived cancers including chronic myeloid leukemia, diffuse large B-cell lymphoma and multiple myeloma,8,13,14 overexpression of Galectin-3 enhanced drug resistance of the TXL2 cells, to both nilotinib and vincristine, whereas TXL2-pMIG-Gal3S6A cells were more sensitive to drug treatment (Figure 2b and c). These results demonstrate that increased intracellular Galectin-3 expression promotes resistance to drug treatment, and that abrogation of its ability to be phosphorylated on S6 results in loss of such capability. Figure 2 Galectin-3 protects ALL cells against drug treatment. (a) TXL2-pMIG, TXL2-pMIG-Gal3 and TXL2-pMIG-Gal3S6A cells cultured without OP9 cells over a 3-day period. Apoptosis was measured using FACS (*P<0.05, **P<0.01). (b, c) TXL2 cells transduced ... To examine the effect of loss of endogenous Galectin-3 on ALL cells, we generated pre-B ALL cells from gal3 +/+ and gal3 −/− BM cells by transduction with Bcr/Abl using standard procedures.15 These cells do not require support from stroma, allowing an assessment of the function of endogenously produced Galectin-3. Transduction efficiency was confirmed by western blot (Supplementary Figure 3). Gal3 +/+ and gal3 −/− pre-B ALL cells had a similar proliferation rate as assessed by cell counting and cell cycle analysis (data not shown). We found that gal3 −/− cells had a specific attenuation of Erk pathway activation, as indicated by a significant decrease in pErk1/2 levels (Figure 2d). To compare drug sensitivity, gal3 +/+ and gal3 −/− pre-B ALL cells were treated with different concentrations of nilotinib or vincristine. As expected, nilotinib or vincristine treatment reduced viability of Bcr/Abl transformed pre-B ALL cells, but gal3 −/− pre-B ALL cells were significantly more sensitive to nilotinib or vincristine treatment than gal3 +/+ pre-B ALL cells (Figure 2e and f). Thus, our studies show that increased levels of Galectin-3, as found in BM plasma and pre-B ALL cells, provide protection to these leukemia cells against drug treatment.

Expansion of multipotent and lymphoid-committed human progenitors through intracellular dimerization of Mpl.

Self-renewal capacity is rapidly lost during differentiation of hematopoietic stem cells to lineage-committed progenitors. We demonstrate here that regulated intracellular signaling through the cytokine receptor Mpl induces profound expansion of not only multipotent (ie, lymphomyeloid) but also lymphoid-committed human hematopoietic progenitors. A fusion protein containing the intracellular signaling domain of Mpl and a dimerization domain was constitutively expressed in populations enriched in human lymphomyeloid progenitor/stem cells (CD34(+)CD38(-)Lin(-)CD7(-)) and multilymphoid progenitors (CD34(+)CD38(-)Lin(-)CD7(+)). Intracellular dimerization of Mpl in target cells was induced by in vitro or in vivo administration of a diffusible synthetic ligand. In vitro, Mpl dimerization produced divisions of clonogenic, multilineage CD34(+) cells able to engraft immunodeficient mice. When dimerization was induced in vivo after transplantation of either lymphomyeloid or multilymphoid progenitors, donor-derived hematopoiesis was sustained for at least 12 weeks and primitive CD34(+)Lin(-) progenitors were expanded more than 1000-fold. Lineage potential of progenitors was not altered and differentiation was not prevented by synthetically induced Mpl signaling. These data demonstrate that dimerization of a single cytokine receptor can deliver a profound expansion signal in both uncommitted and lymphoid-committed human hematopoietic progenitors.