A novel approach for the identification of efficient combination therapies in primary human acute myeloid leukemia specimens.

Appropriate culture methods for the interrogation of primary leukemic samples were hitherto lacking and current assays for compound screening are not adapted for large-scale investigation of synergistic combinations. In this study, we report a novel approach that efficiently distills synthetic lethal interactions between small molecules active on primary human acute myeloid leukemia (AML) specimens. In single-dose experiments and under culture conditions preserving leukemia stem cell activity, our strategy considerably reduces the number of tests needed for the identification of promising compound combinations. Initially conducted with a selected library of 5000 small molecules and 20 primary AML specimens, it reveals 5 broad classes of sensitized therapeutic target pathways along with their synergistic patient-specific fingerprints. This novel method opens new avenues for the development of AML personalized therapeutics and may be generalized to other tumor types, for which in vitro cancer stem cell cultures have been developed.

Transcriptome analysis of G protein-coupled receptors in distinct genetic subgroups of acute myeloid leukemia: identification of potential disease-specific targets.

Acute myeloid leukemia (AML) is associated with poor clinical outcome and the development of more effective therapies is urgently needed. G protein-coupled receptors (GPCRs) represent attractive therapeutic targets, accounting for approximately 30% of all targets of marketed drugs. Using next-generation sequencing, we studied the expression of 772 GPCRs in 148 genetically diverse AML specimens, normal blood and bone marrow cell populations as well as cord blood-derived CD34-positive cells. Among these receptors, 30 are overexpressed and 19 are downregulated in AML samples compared with normal CD34-positive cells. Upregulated GPCRs are enriched in chemokine (CCR1, CXCR4, CCR2, CX3CR1, CCR7 and CCRL2), adhesion (CD97, EMR1, EMR2 and GPR114) and purine (including P2RY2 and P2RY13) receptor subfamilies. The downregulated receptors include adhesion GPCRs, such as LPHN1, GPR125, GPR56, CELSR3 and GPR126, protease-activated receptors (F2R and F2RL1) and the Frizzled family receptors SMO and FZD6. Interestingly, specific deregulation was observed in genetically distinct subgroups of AML, thereby identifying different potential therapeutic targets in these frequent AML subgroups.

Favorable long-term outcome of patients with multiple myeloma using a frontline tandem approach with autologous and non-myeloablative allogeneic transplantation.

Despite survival improvement with novel agents and use of autologous hematopoietic stem cell transplantation (HSCT), cure of patients with multiple myeloma (MM) remains anecdotal. Initial observations suggested that chronic GvHD was accompanied by an anti-myeloma effect after myeloablative HSCT, but unfortunately this procedure was hampered by high non-relapse mortality (NRM). To maximize the anti-myeloma effect and minimize NRM, we developed a non-myeloablative (NMA) regimen associated with a high incidence of chronic GvHD and tested its efficacy on patient survival and disease eradication. From 2001 to 2010, 92 patients aged ⩽ 65 years with a compatible sibling donor received autologous HSCT followed by an outpatient NMA allogeneic HSCT using a conditioning of fludarabine and cyclophosphamide. Patient median age was 52 years and 97% presented Durie-Salmon stages II-III disease. After a median follow-up of 8.8 years, probability of 10-year progression free and overall survival were 41% and 62%, respectively. Although the cumulative incidence of extensive chronic GvHD was high (at 79%), the majority of long-term survivors were off immunosuppressive drugs by year 5 and NRM was low (at 10%). Together, our results suggest that potential MM cure can be achieved with NMA transplantation regimens that maximize graft-versus-myeloma effect and minimize NRM.

Disclosure of incidental findings in cancer genomic research: investigators' perceptions on obligations and barriers.

Although there has been significant research surrounding incidental findings (IFs), the guidelines and information provided to investigators remain unspecific, unclear, and often generalize the course of action to everyone in the field. We explored the perceptions and experiences of investigators regarding the return of IFs in genetic research. Researchers and clinician-researchers were invited to participate in semi-structured telephone interviews in Quebec and Ontario. Twenty professionals participated, and thematic analysis was used to analyze the transcriptions. Four contextual elements emerged: (i) degree of significance of results, (ii) respect for persons, (iii) infrastructure implications, and (iv) professional responsibilities. Our findings demonstrate that all investigators raised similar contextual elements surrounding the return of IFs. However, some nuances in participants' experiences of the understanding of professional responsibilities also emerged. Because of the existing nuances, a one-size-fits-all approach is inappropriate, suggesting that context ought to be considered in decisions about IFs.

Epigenetic regulation of GATA2 and its impact on normal karyotype acute myeloid leukemia.

The GATA2 gene encodes a zinc-finger transcription factor that acts as a master regulator of normal hematopoiesis. Mutations in GATA2 have been implicated in the development of myelodysplastic syndrome and acute myeloid leukemia (AML). Using RNA sequencing we now report that GATA2 is either mutated with a functional consequence, or expressed at low levels in the majority of normal karyotype AML (NK-AML). We also show that low-GATA2-expressing specimens (GATA2(low)) exhibit allele-specific expression (ASE) (skewing) in more than half of AML patients examined. We demonstrate that the hypermethylation of the silenced allele can be reversed by exposure to demethylating agents, which also restores biallelic expression of GATA2. We show that GATA2(low) AML lack the prototypical R882 mutation in DNMT3A frequently observed in NK-AML patients and that The Cancer Genome Atlas AML specimens with DNMT3A R882 mutations are characterized by CpG hypomethylation of GATA2. Finally, we validate that several known missense single-nucleotide polymorphisms in GATA2 are actually loss-of-function variants, which, when combined with ASE, represent the equivalent of homozygous GATA2 mutations. From a broader perspective, this work suggests for the first time that determinants of ASE likely have a key role in human leukemia.

Funding considerations for the disclosure of genetic incidental findings in biobank research.

The use of biobanks in biomedical research has grown considerably in recent years. As a result of the increasing analysis of tissue samples stored in biobanks, there has also been an increase in the probability of discovering-in addition to the research target-incidental findings (IF). We identified 23 laws, policies and guidelines from international, regional and national organizations that provide guidance or identify the need for the disclosure of IF to research participants. We analyzed these instruments to determine their contemplation of the funding considerations for the disclosure of IF, examining their guidance for who discloses and the extent of researcher responsibilities. We found that the available normative documents provide little guidance to researchers and biobanks for how they should address cost and funding concerns associated with IF disclosure. It is therefore essential that the research and policy communities think through the financial implications of imposing an ethical responsibility to disclose IF. Concerted efforts should be made by policymakers, ethicists, researchers, clinicians and research institutions to develop detailed funding recommendations, potentially universal in application, to aid in the disclosure of IF, and we provide recommendations on steps that can be taken to ensure full consideration of these issues.

Complementary and independent function for Hoxb4 and Bmi1 in HSC activity.

Determinants regulating short- and long-term repopulating hematopoietic stem cell (STR-HSC and LTR-HSC) self-renewal remain largely uncharacterized. To gain further insights into HSC self-renewal, we investigated possible genetic interactions between two well-recognized regulators of this process: Bmi1 and Hoxb4. Using complementation and overexpression strategies in mouse HSCs, we document that Bmi1 is not required for the in vivo expansion of fetal HSCs but is essential for the long-term maintenance of adult HSCs. Importantly, we show that Hoxb4 overexpression induces an expansion of Bmi1(-/-)STR-HSCs leading to a rescue of their repopulation defect. In contrast to Hoxb4, we also show that Bmi1 fails to induce HSC expansion ex vivo. Consistent with these results, we report high levels of Angptl3 and Cbx7 in Hoxb4- and Bmi1-transduced cells, respectively. Together, these results support the emerging concept that fate and sustainability of this fate are two critical components of self-renewal in adult stem cells such as HSCs.

Evidence for Hox and E2A-PBX1 collaboration in mouse T-cell leukemia.

Using murine Moloney leukemia virus (MMLV)-based proviral insertional mutagenesis, we previously showed a preferential targeting of a small region in the Hoxa gene locus in E2A-PBX1-induced lymphoid leukemia resulting in the overexpression of several Hoxa genes including Hoxa10, Hoxa9 and Hoxa7. This observation suggested a functional interaction between Hox gene overexpression and E2A-PBX1 in lymphoid tumors. To further explore this possibility, we generated a series of compound E2A-PBX1 x Hox transgenic mice and tested the genetic interaction between these genes in the generation of lymphoid leukemia in vivo. Results presented in this report show that the onset of T-cell leukemia is significantly accelerated in E2A-PBX1 x Hoxb4 compound transgenic animals when compared with control E2A-PBX1 or Hoxb4 littermates. Hoxa9 appears less potent than Hoxb4 to accelerate E2A-PBX1-induced T-cell leukemia in mice. E2A-PBX1-induced T-cell leukemias express much higher levels of Hoxa genes than MMLV-induced counterparts, possibly suggesting a contribution of these genes to T-cell transformation by E2A-PBX1. Collectively, these data provide the first genetic evidence showing oncogenic collaboration between E2A-PBX1 and a Hox gene in lymphoid malignancies in vivo and document the specific deregulation of a subgroup of Hoxa genes in these leukemias.

HOXB4 overexpression mediates very rapid stem cell regeneration and competitive hematopoietic repopulation.

OBJECTIVE: Hox transcription factors have emerged as important regulators of hematopoiesis. In particular, we have shown that overexpression of HOXB4 in mouse bone marrow can greatly enhance the level of hematopoietic stem cell (HSC) regeneration achieved at late times (> 4 months) posttransplantation. The objective of this study was to resolve if HOXB4 increases the rate and/or duration of HSC regeneration, and also to see if this enhancement was associated with impaired production of end cells or would lead to competitive reconstitution of all compartments. METHODS: Retroviral vectors were generated with the GFP reporter gene +/- HOXB4 to enable the isolation and direct tracking of transduced cells in culture or following transplantation. Stem cell recovery was measured by limit dilution assay for long-term competitive repopulating cells (CRU). RESULTS: HOXB4-overexpressing cells have enhanced growth in vitro, as demonstrated by their rapid dominance in mixed cultures and their shortened population doubling time. Furthermore, HOXB4-transduced cells have a marked competitive repopulating advantage in vivo in both primitive and mature compartments. CRU recovery in HOXB4 recipients was extremely rapid, reaching 25% of normal by 14 days posttransplant or some 80-fold greater than control transplant recipients, and attaining normal numbers by 12 weeks. Mice transplanted with even higher numbers of HOXB4-transduced CRU regenerated up to but not beyond the normal CRU levels. CONCLUSIONS: HOXB4 is a potent enhancer of primitive hematopoietic cell growth, likely by increasing self-renewal probability but without impairing homeostatic control of HSC population size or the rate of production and maintenance of mature end cells.

NUP98-HOXA9 expression in hemopoietic stem cells induces chronic and acute myeloid leukemias in mice.

Here we describe hemopoietic chimeras serving as a mouse model for NUP98-HOXA9-induced leukemia, which reproduced several of the phenotypes observed in human disease. Mice transplanted with bone marrow cells expressing NUP98-HOXA9 through retroviral transduction acquire a myeloproliferative disease (MPD) and eventually succumb to acute myeloid leukemia (AML). The NUP98 portion of the fusion protein was shown to be responsible for transforming a clinically silent pre-leukemic phase observed for Hoxa9 into a chronic, stem cell-derived MPD. The co-expression of NUP98-HOXA9 and Meis1 accelerated the transformation of MPD to AML, identifying a genetic interaction previously observed for Hoxa9 and Meis1. Our findings demonstrate the presence of overlapping yet distinct molecular mechanisms for MPD versus AML, illustrating the complexity of leukemic transformation.

Defining roles for HOX and MEIS1 genes in induction of acute myeloid leukemia.

Complex genetic and biochemical interactions between HOX proteins and members of the TALE (i.e., PBX and MEIS) family have been identified in embryonic development, and some of these interactions also appear to be important for leukemic transformation. We have previously shown that HOXA9 collaborates with MEIS1 in the induction of acute myeloid leukemia (AML). In this report, we demonstrate that HOXB3, which is highly divergent from HOXA9, also genetically interacts with MEIS1, but not with PBX1, in generating AML. In addition, we show that the HOXA9 and HOXB3 genes play key roles in establishing all the main characteristics of the leukemias, while MEIS1 functions only to accelerate the onset of the leukemic transformation. Contrasting the reported functional similarities between PREP1 and MEIS1, such as PBX nuclear retention, we also show that PREP1 overexpression is incapable of accelerating the HOXA9-induced AML, suggesting that MEIS1 function in transformation must entail more than PBX nuclear localization. Collectively, these data demonstrate that MEIS1 is a common leukemic collaborator with two structurally and functionally divergent HOX genes and that, in this collaboration, the HOX gene defines the identity of the leukemia.

Allogeneic transplantation for multiple myeloma: further evidence for a GVHD-associated graft-versus-myeloma effect.

We report a series of 37 consecutive patients with multiple myeloma (MM) who received an allograft between 1990 and 2000 at our institution. Median age was 47 years, and nearly 70% of patients were Durie-Salmon stage III. A median of five cycles of chemotherapy were given before transplant, with a median interval between diagnosis and transplant of 9.3 months. We report a nonrelapse mortality rate of 22% with a median follow-up period of 40 months, whereas complete remission (CR) rate at 12 months is estimated at 57%. Treatment failure rate and overall survival at 40 months are estimated at 52% and 32%, respectively. The number of chemotherapy cycles prior to allotransplantation achieved borderline statistical significance as a poor prognosis factor for overall survival (P = 0.05), while the presence of chronic graft-versus-host disease (cGVHD) was significantly correlated with CR achievement (P = 0.036). Our study confirms that early allografting in MM can yield toxicity rates significantly lower than those associated with historical cohorts, and supports the hypothesis that cumulative chemotoxicity has a negative influence on mortality and survival rates. More importantly, our study clearly demonstrates an association between cGVHD and CR and brings further evidence in favor of a graft-versus-myeloma effect.

AP-1 complex is effector of Hox-induced cellular proliferation and transformation.

Hox gene products, initially characterized as master regulators of embryonic patterning, are also required for proper functioning of adult tissues. There is also a growing body of evidence that links Hox proteins to regulation of cellular proliferation/transformation. However, the underlying molecular mechanisms of Hox-associated transformation and tissue growth have yet to be elucidated. Using a well established model system for studying changes in cellular proliferation induced by Hoxb4, we show that AP-1 activity is markedly increased in Hoxb4-transduced cells due to significant upregulation of Jun-B and Fra-1 protein levels. Furthermore, we also show that the specific changes in AP-1 protein expression are necessary for the proliferation effects induced by Hoxb4, and that these changes converge to increase levels of cyclin D1, a known integrator of proliferation signals. Our observations thus link Hox gene products with key elements of the cell cycle machinery.

Enhanced in vivo regenerative potential of HOXB4-transduced hematopoietic stem cells with regulation of their pool size.

After bone marrow transplantation (BMT), there is a rapid regeneration to normal pretransplantation levels in the number of hematopoietic progenitors and mature end cells, whereas hematopoietic stem cell (HSC) numbers recover to only 5% to 10% of normal levels. This suggests that HSC are significantly restricted in their self-renewal behavior and hence in their ability to repopulate the host stem cell compartment. Previously, we have reported that HSC engineered to overexpress the homeobox transcription factor HOXB4 have a large repopulation advantage over untransduced cells as assessed at 4 months in a murine transplantation model (Sauvageau et al, Genes Dev 9:1753, 1995). This phenomenon has now been examined in detail for periods extending to 12 months in cohorts of mice transplanted with various numbers of HOXB4-transduced HSC. In all mice analyzed, HOXB4-transduced HSC were capable of fully reconstituting the HSC compartment, resulting, on average, in some 14-fold greater numbers of HSC than observed when transplanting control, non-HOXB4-transduced bone marrow cells. These data indicate that HOXB4 is a limiting factor in the regeneration of HSC to normal levels after BMT. Furthermore, we show that HOXB4-transduced HSC did not expand above levels normally observed in unmanipulated mice, indicating that its overexpression does not override the regulatory mechanisms that maintain the HSC pool size within normal limits.

Functional antagonism of the Polycomb-Group genes eed and Bmi1 in hemopoietic cell proliferation.

The murine Polycomb-Group (PcG) proteins Eed and Bmi1 govern axial patterning during embryonic development by segment-specific repression of Hox gene expression. The two proteins engage in distinct multimeric complexes that are thought to use a common molecular mechanism to render the regulatory regions of Hox and other downstream target genes inaccessible to transcriptional activators. Beyond axial patterning, Bmi1 is also involved in hemopoiesis because a loss-of-function allele causes a profound decrease in bone marrow progenitor cells. Here, evidence is presented that is consistent with an antagonistic function of eed and Bmi1 in hemopoietic cell proliferation. Heterozygosity for an eed null allele causes marked myelo- and lymphoproliferative defects, indicating that eed is involved in the negative regulation of the pool size of lymphoid and myeloid progenitor cells. This antiproliferative function of eed does not appear to be mediated by Hox genes or the tumor suppressor locus p16(INK4a)/p19(ARF) because expression of these genes was not altered in eed mutants. Intercross experiments between eed and Bmi1 mutant mice revealed that Bmi1 is epistatic to eed in the control of primitive bone marrow cell proliferation. However, the genetic interaction between the two genes is cell-type specific as the presence of one or two mutant alleles of eed trans-complements the Bmi1-deficiency in pre-B bone marrow cells. These studies thus suggest that hemopoietic cell proliferation is regulated by the relative contribution of repressive (Eed-containing) and enhancing (Bmi1-containing) PcG gene complexes.