Systems medicine dissection of chr1q-amp reveals a novel PBX1-FOXM1 axis for targeted therapy in multiple myeloma.

Understanding the biological and clinical impact of copy number aberrations (CNAs) on the development of precision therapies in cancer remains an unmet challenge. Genetic amplification of chromosome 1q (chr1q-amp) is a major CNA conferring an adverse prognosis in several types of cancer, including in the blood cancer multiple myeloma (MM). Although several genes across chromosome 1 (chr1q) portend high-risk MM disease, the underpinning molecular etiology remains elusive. Here, with reference to the 3-dimensional (3D) chromatin structure, we integrate multi-omics data sets from patients with MM with genetic variables to obtain an associated clinical risk map across chr1q and to identify 103 adverse prognosis genes in chr1q-amp MM. Prominent among these genes, the transcription factor PBX1 is ectopically expressed by genetic amplification and epigenetic activation of its own preserved 3D regulatory domain. By binding to reprogrammed superenhancers, PBX1 directly regulates critical oncogenic pathways and a FOXM1-dependent transcriptional program. Together, PBX1 and FOXM1 activate a proliferative gene signature that predicts adverse prognosis across multiple types of cancer. Notably, pharmacological disruption of the PBX1-FOXM1 axis with existing agents (thiostrepton) and a novel PBX1 small molecule inhibitor (T417) is selectively toxic against chr1q-amp myeloma and solid tumor cells. Overall, our systems medicine approach successfully identifies CNA-driven oncogenic circuitries, links them to clinical phenotypes, and proposes novel CNA-targeted therapy strategies in MM and other types of cancer.

Chromatin-based, in cis and in trans regulatory rewiring underpins distinct oncogenic transcriptomes in multiple myeloma.

Multiple myeloma is a genetically heterogeneous cancer of the bone marrow plasma cells (PC). Distinct myeloma transcriptome profiles are primarily driven by myeloma initiating events (MIE) and converge into a mutually exclusive overexpression of the CCND1 and CCND2 oncogenes. Here, with reference to their normal counterparts, we find that myeloma PC enhanced chromatin accessibility combined with paired transcriptome profiling can classify MIE-defined genetic subgroups. Across and within different MM genetic subgroups, we ascribe regulation of genes and pathways critical for myeloma biology to unique or shared, developmentally activated or de novo formed candidate enhancers. Such enhancers co-opt recruitment of existing transcription factors, which although not transcriptionally deregulated per se, organise aberrant gene regulatory networks that help identify myeloma cell dependencies with prognostic impact. Finally, we identify and validate the critical super-enhancer that regulates ectopic expression of CCND2 in a subset of patients with MM and in chronic lymphocytic leukemia.

High resolution IgH repertoire analysis reveals fetal liver as the likely origin of life-long, innate B lymphopoiesis in humans.

The ontogeny of the natural, public IgM repertoire remains incompletely explored. Here, high-resolution immunogenetic analysis of B cells from (unrelated) fetal, child, and adult samples, shows that although fetal liver (FL) and bone marrow (FBM) IgM repertoires are equally diversified, FL is the main source of IgM natural immunity during the 2nd trimester. Strikingly, 0.25% of all prenatal clonotypes, comprising 18.7% of the expressed repertoire, are shared with the postnatal samples, consistent with persisting fetal IgM+ B cells being a source of natural IgM repertoire in adult life. Further, the origins of specific stereotypic IgM+ B cell receptors associated with chronic lymphocytic leukemia, can be traced back to fetal B cell lymphopoiesis, suggesting that persisting fetal B cells can be subject to malignant transformation late in life. Overall, these novel data provide unique insights into the ontogeny of physiological and malignant B lymphopoiesis that spans the human lifetime.

Activated invariant NKT cells regulate osteoclast development and function.

Invariant NKT (iNKT) cells modulate innate and adaptive immune responses through activation of myeloid dendritic cells and macrophages and via enhanced clonogenicity, differentiation, and egress of their shared myeloid progenitors. Because these same progenitors give rise to osteoclasts (OCs), which also mediate the egress of hematopoietic progenitors and orchestrate bone remodeling, we hypothesized that iNKT cells would extend their myeloid cell regulatory role to the development and function of OCs. In this study, we report that selective activation of iNKT cells by α-galactosylceramide causes myeloid cell egress, enhances OC progenitor and precursor development, modifies the intramedullary kinetics of mature OCs, and enhances their resorptive activity. OC progenitor activity is positively regulated by TNF-α and negatively regulated by IFN-γ, but is IL-4 and IL-17 independent. These data demonstrate a novel role of iNKT cells that couples osteoclastogenesis with myeloid cell egress in conditions of immune activation.

Pulmonary, gonadal, and central nervous system status after bone marrow transplantation for sickle cell disease.

We conducted a prospective, multicenter investigation of human-leukocyte antigen (HLA) identical sibling bone marrow transplantation (BMT) in children with severe sickle cell disease (SCD) between 1991 and 2000. To determine if children were protected from complications of SCD after successful BMT, we extended our initial study of BMT for SCD to conduct assessments of the central nervous system (CNS) and of pulmonary function 2 or more years after transplantation. In addition, the impact on gonadal function was studied. After BMT, patients with stroke who had stable engraftment of donor cells experienced no subsequent stroke events after BMT, and brain magnetic resonance imaging (MRI) exams demonstrated stable or improved appearance. However, 2 patients with graft rejection had a second stroke after BMT. After transplantation, most patients also had unchanged or improved pulmonary function. Among the 11 patients who had restrictive lung changes at baseline, 5 were improved and 6 had persistent restrictive disease after BMT. Of the 2 patients who had obstructive changes at baseline, 1 improved and 1 had worsened obstructive disease after BMT. There was, however, significant gonadal toxicity after BMT, particularly among female recipients. In summary, individuals who had stable donor engraftment did not experience sickle-related complications after BMT, and were protected from progressive CNS and pulmonary disease.

Regulation of hematopoiesis in vitro and in vivo by invariant NKT cells.

Invariant natural killer T cells (iNKT cells) are a small subset of immunoregulatory T cells highly conserved in humans and mice. On activation by glycolipids presented by the MHC-like molecule CD1d, iNKT cells promptly secrete T helper 1 and 2 (Th1/2) cytokines but also cytokines with hematopoietic potential such as GM-CSF. Here, we show that the myeloid clonogenic potential of human hematopoietic progenitors is increased in the presence of glycolipid-activated, GM-CSF-secreting NKT cells; conversely, short- and long-term progenitor activity is decreased in the absence of NKT cells, implying regulation of hematopoiesis in both the presence and the absence of immune activation. In accordance with these findings, iNKT-cell-deficient mice display impaired hematopoiesis characterized by peripheral-blood cytopenias, reduced marrow cellularity, lower frequency of hematopoietic stem cells (HSCs), and reduced early and late hematopoietic progenitors. We also show that CD1d is expressed on human HSCs. CD1d-expressing HSCs display short- and long-term clonogenic potential and can present the glycolipid alpha-galactosylceramide to iNKT cells. Thus, iNKT cells emerge as the first subset of regulatory T cells that are required for effective hematopoiesis in both steady-state conditions and under conditions of immune activation.

Human invariant NKT cells are required for effective in vitro alloresponses.

NKT cells are a small subset of regulatory T cells conserved in humans and mice. In humans they express the Valpha24Jalpha18 invariant chain (hence invariant NKT (iNKT) cells) and are restricted by the glycolipid-presenting molecule CD1d. In mice, iNKT cells may enhance or inhibit anti-infectious and antitumor T cell responses but suppress autoimmune and alloreactive responses. We postulated that iNKT cells might also modulate human alloreactive responses. Using MLR assays we demonstrate that in the presence of the CD1d-presented glycolipid alpha-galactosylceramide (alphaGC) alloreactivity is enhanced (37 +/- 12%; p < 0.001) in an iNKT cell-dependent manner. iNKT cells are activated early during the course of the MLR, presumably by natural ligands. In MLR performed without exogenous ligands, depletion of iNKT cells significantly diminished the alloresponse in terms of proliferation (58.8 +/- 24%; p < 0.001) and IFN-gamma secretion (43.2 +/- 15.2%; p < 0.001). Importantly, adding back fresh iNKT cells restored the reactivity of iNKT cell-depleted MLR to near baseline levels. CD1d-blocking mAbs equally reduced the reactivity of the iNKT cell-replete and -depleted MLR compared with IgG control, indicating that the effect of iNKT cells in the in vitro alloresponse is CD1d-dependent. These findings suggest that human iNKT cells, although not essential for its development, can enhance the alloreactive response.

Can routine commercial cord blood banking be scientifically and ethically justified?

BACKGROUND TO THE DEBATE: Umbilical cord blood--the blood that remains in the placenta after birth--can be collected and stored frozen for years. A well-accepted use of cord blood is as an alternative to bone marrow as a source of hematopoietic stem cells for allogeneic transplantation to siblings or to unrelated recipients; women can donate cord blood for unrelated recipients to public banks. However, private banks are now open that offer expectant parents the option to pay a fee for the chance to store cord blood for possible future use by that same child (autologous transplantation).

Microchimerism in female bone marrow and bone decades after fetal mesenchymal stem-cell trafficking in pregnancy.

Fetal cells enter maternal blood during pregnancy and persist in women with autoimmune disease. The frequency of subsequent fetomaternal microchimerism in healthy women and its cell type is unknown. To test the hypothesis that fetal mesenchymal stem cells persist in maternal organs, we studied female bone marrow and ribs. Male cells were identified by XY fluorescence in-situ hybridisation in marrow-derived mesenchymal stem cells and in rib sections from all women with male pregnancies, but not in controls (9/9 vs 0/5, p=0.0005). We conclude that fetal stem cells transferred into maternal blood engraft in marrow, where they remain throughout life. This finding has implications for normal pregnancy, for obstetric complications that increase fetomaternal trafficking, and for graft survival after transplantation.

CD34+ cells from first-trimester fetal blood are enriched in primitive hemopoietic progenitors.

OBJECTIVE: The purpose of this study was to investigate whether purified CD34(+) cells from first-trimester fetal blood are a source of primitive and committed hemopoietic progenitors. STUDY DESIGN: CD34(+) cells from first-trimester fetal blood and term cord blood were assayed for committed hemopoietic progenitor cells, high proliferative potential colony-forming cells, and long-term culture-initiating cells. RESULTS: First-trimester CD34(+) cells that were compared with cells at term generated fewer hemopoietic progenitor cells and fewer high proliferative potential colony-forming cells with lower recloning efficiency(P <.001). First-trimester CD34(+) cells tended to contain more long-term culture-initiating cells, both in bulk cultures and by limiting dilution analysis. The ratio between committed and primitive progenitors was 3 in the first-trimester and 20 in the term cord blood, respectively. CONCLUSION: First-trimester fetal blood is enriched in primitive (compared with committed) hemopoietic progenitors and may be an advantageous source of stem cells for prenatal therapy.

Stem cell transplantation for chronic myeloid leukemia in children.

Hematopoietic stem cell transplantation (SCT) is the only proven cure for chronic myeloid leukemia (CML), a rare disease in childhood. We report outcomes of 314 children with Philadelphia-chromosome-positive (Ph+) CML undergoing SCT from HLA-matched siblings (n = 182) or volunteer-unrelated donors (VUD; n = 132). Three-year overall survival (OS) and leukemia-free survival (LFS) rates were 66% and 55% (n = 314). For 156 children in first chronic phase (CP1) who underwent transplantation from HLA-identical siblings, OS and LFS rates were 75% and 63%. For 97 children who underwent SCT in CP1 from VUD, 3-year OS and LFS rates were 65% and 56%, reflecting higher transplantation-related mortality (TRM) after VUD SCT (35% vs 20%; multivariate hazard ratio [HR], 1.9; 95% confidence interval [CI], 1.0-3.5; P =.05). In a multivariate model for OS and LFS, outcomes were superior in CP1 than in advanced phase (AP/CP1) (OS HR, 2.0; 95% CI, 1.3-3; P =.001; LFS HR, 1.8; 95% CI, 1.2-2.6; P =.003). For relapse, donor source (VUD/sibling) (HR, 0.38; 95% CI, 0.19-0.76; P =.006) and disease stage (AP/CP1) (HR, 2.4; 95% CI, 1.36-4.3; P =.003) were significant. This is the first large series to show that SCT confers long-term LFS in most children with CML and helps assess alternative therapy, including tyrosine kinase inhibitors.

Bone marrow transplantation for beta-thalassaemia major: the UK experience in two paediatric centres.

Stem cell transplantation (SCT) remains the only cure for thalassaemia major. Recent advances in medical treatment make it even more important that accurate information is available regarding outcome of SCT in relevant patient populations in order to guide informed decisions regarding the most appropriate treatment for individual thalassaemia patients. We report the results of 55 consecutive first related allogeneic bone marrow transplants (BMT) for children with beta-thalassaemia major performed in two UK paediatric centres over 10 years. Between February 1991 and February 2001, 55 children underwent 57 allogeneic BMT. The median age at BMT was 6.4 years and the majority of patients (73%) originated from the Indian subcontinent. Using the Pesaro risk classification, 17 patients were class 1, 27 were class 2 and 11 were class 3. Actuarial overall survival and thalassaemia-free survival at 8 years were 94.5% (95% CI 85.1-98.1) and 81.8% (95% CI 69.7-89.8) respectively. Despite the majority of patients being in class 2 or 3, transplant-related mortality was low (5.4%). The principal complication was graft rejection accompanied by autologous reconstitution that occurred in 13.2% of transplants. Following modification of the conditioning regimen in 1993, the rejection rate fell to 4.6% and remained low. Acute graft-versus-host disease (GVHD) of grade II-IV occurred in 31% and chronic GVHD in 14.5%. These data compare favourably with survival with medical treatment for thalassaemia major and suggest that allogeneic BMT remains an important treatment option for children with beta-thalassaemia major, particularly when compliance with iron chelation is poor.

Expandability of haemopoietic progenitors in first trimester fetal and maternal blood: implications for non-invasive prenatal diagnosis.

OBJECTIVES: Selective amplification of rare fetal cells in maternal blood is a potential strategy for non-invasive prenatal diagnosis. We assessed the proliferative potential of first trimester fetal progenitors compared to maternal ones. METHODS: Fetal and maternal haemopoietic progenitors were cultured separately and in two model mixtures: (i) co-cultures of male fetal nucleated cells mixed with maternal nucleated cells and (ii) co-cultures of malefetal CD34+ cells with maternal CD34+ cells. Cell origin was detected by X-Y fluorescence in situ hybridisation (FISH) RESULTS: The frequency of haemopoietic progenitors in first trimester fetal blood (predominantly CFU-GEMM) differed from those in peripheral blood from pregnant women (predominantly BFU-e). First trimester haemopoietic progenitors formed larger colonies (p=0.0001) and their haemoglobinisation was accelerated compared to those of maternal origin (p<0.001). CD34+ fetal haemopoietic progenitor cells could be expanded four times more than their maternal counterparts (median 235.8-fold, range 174.0-968.0 vs 71.9-fold, range 41.1-192.0; p=0.003). While selective expansion of fetal cells was not observed in the mononuclear cell model, the CD34+ cell rare event mixtures produced a 463.2-fold (range 128.0-2915.0) expansion of fetal cells. CONCLUSION: Selective expansion of first trimester fetal haemopoietic progenitors may be useful for amplifying fetal cells from maternal blood.