Mary Ellen Martin

MLL Targets SET Domain Methyltransferase Activity to Hox Gene Promoters

MLL, the human homolog of Drosophila trithorax, maintains Hox gene expression in mammalian embryos and is rearranged in human leukemias resulting in Hox gene deregulation. How MLL or MLL fusion proteins regulate gene expression remains obscure. We show that MLL regulates target Hox gene expression through direct binding to promoter sequences. We further show that the MLL SET domain is a histone H3 lysine 4-specific methyltransferase whose activity is stimulated with acetylated H3 peptides. This methylase activity is associated with Hox gene activation and H3 (Lys4) methylation at cis-regulatory sequences in vivo. A leukemogenic MLL fusion protein that activates Hox expression had no effect on histone methylation, suggesting a distinct mechanism for gene regulation by MLL and MLL fusion proteins.

Hoxa9 and Meis1 Are Key Targets for MLL-ENL-Mediated Cellular Immortalization

ABSTRACT MLL fusion proteins are oncogenic transcription factors that are associated with aggressive lymphoid and myeloid leukemias. We constructed an inducible MLL fusion, MLL-ENL-ERtm, that rendered the transcriptional and transforming properties of MLL-ENL strictly dependent on the presence of 4-hydroxy-tamoxifen. MLL-ENL-ERtm-immortalized hematopoietic cells required 4-hydroxy-tamoxifen for continuous growth and differentiated terminally upon tamoxifen withdrawal. Microarray analysis performed on these conditionally transformed cells revealed Hoxa9 and Hoxa7 as well as the Hox coregulators Meis1 and Pbx3 among the targets upregulated by MLL-ENL-ERtm. Overexpression of the Hox repressor Bmi-1 inhibited the growth-transforming activity of MLL-ENL. Moreover, the enforced expression of Hoxa9 in combination with Meis1 was sufficient to substitute for MLL-ENL-ERtm function and to maintain a state of continuous proliferation and differentiation arrest. These results suggest that MLL fusion proteins impose a reversible block on myeloid differentiation through aberrant activation of a limited set of homeobox genes and Hox coregulators that are consistently expressed in MLL-associated leukemias.

Dimerization of MLL fusion proteins immortalizes hematopoietic cells

MLL fusion proteins are leukemogenic, but their mechanism is unclear. Induced dimerization of a truncated MLL immortalizes bone marrow and imposes a reversible block on myeloid differentiation associated with upregulation of Hox a7, a9, and Meis1. Both dimerized MLL and exon-duplicated MLL are potent transcriptional activators, suggesting a link between dimerization and partial tandem duplication of DNA binding domains of MLL. Dimerized MLL binds with higher affinity than undimerized MLL to a CpG island within the Hox a9 locus. However, MLL-AF9 is not dimerized in vivo. The data support a model in which either MLL dimerization/exon duplication or fusion to a transcriptional activator results in Hox gene upregulation and ultimately transformation.

Leukemogenic MLL Fusion Proteins Bind across a Broad Region of the Hox a9 Locus, Promoting Transcription and Multiple Histone Modifications

Chromosome translocations involving the mixed lineage leukemia gene MLL are associated with aggressive acute leukemias in both children and adults. Leukemogenic MLL fusion proteins delete the MLL SET domain Lys(4) methyltransferase activity and fuse MLL to 1 of >40 different translocation partners. Some MLL fusion proteins involve nuclear proteins that are transcriptional activators, whereas others have transcriptional activating activity but instead dimerize the truncated MLL molecule. Both types of MLL fusion proteins enforce persistent expression of Hox a9 and Meis1, which is pivotal for leukemogenesis through mechanisms that remain obscure. Here, we show that nuclear and dimerizable forms of MLL bind with a similar pattern to the Hox a9 locus that overlaps the distribution of wild-type MLL and deregulate transcription of three isoforms of Hox a9. Induction of MLL fusion protein activity is associated with increased levels of histone acetylation and Lys(4) methylation at Hox target genes. In addition, the MLL-ENL-ER protein, but not dimerized MLL, also induces dimethylation of histone H3 at Lys(79), suggesting alternative mechanisms for transcriptional activation.

Lack of effect of donor-recipient ABO mismatching on outcome following allogeneic hematopoietic stem cell transplantation.

Several recently published studies have suggested that patients who undergo ABO mismatched hematopoietic stem cell transplantation may be at increased risk for relapse, graft-versus-host disease, transplant-related mortality, and/or all-cause mortality. To investigate this issue further, we analyzed potential associations between the donor–recipient ABO mismatch pattern and the above outcome measures among 240 consecutive patients who underwent allogeneic hematopoietic stem cell transplantation at our institution. Our analyses uncovered no significant associations between donor–recipient ABO mismatch pattern and overall survival, event-free survival, transplant-related mortality, incidence of acute graft-versus-host disease (GVHD), or incidence of chronic GVHD. Our data do not support recent assertions that donor–recipient ABO mismatching is a major risk factor for patients undergoing allogeneic transplant, nor do they support recent assertions that ABO matching should be an important consideration in selecting allogeneic hematopoietic stem cell donors.

Allogeneic hematopoietic SCT for primary cutaneous T cell lymphomas

Cutaneous T-cell lymphomas (CTCL) are a heterogeneous group of non-Hodgkin lymphomas that are considered incurable. The role of allogeneic hematopoietic SCT (HSCT) in the treatment of CTCL is not well defined but may provide potent graft-vs-lymphoma (GVL) activity independent of the conditioning therapy. We present outcomes of 12 extensively-pretreated patients with CTCL who underwent allogeneic HSCT using, most commonly, a reduced intensity conditioning regimen. Median age at diagnosis of CTCL was 49 years, and median time to transplantation from diagnosis was 3.3 years. Transplantation induced and maintained CR in six patients with active disease, supporting the presence of a GVL effect. TRM was low, and 42% of patients were alive and disease-free a median duration of 22 months after transplant. Two patients showed strong and direct evidence of a GVL-effect with a direct response to withdrawal of immunosuppression or to donor leukocyte infusion. Our data show that HSCT can provide long-term disease control in patients with advanced CTCL, which otherwise was refractory to immunotherapy and chemotherapy.

Lack of effect of donor‐recipient Rh mismatch on outcomes after allogeneic hematopoietic stem cell transplantation

BACKGROUND: A recently published study has reported that donor‐recipient Rhesus (Rh)‐mismatched allogeneic hematopoietic stem cell transplantation independently led to significantly poorer survival. This suggests that donor‐recipient Rh mismatching is a risk factor in allogeneic hematopoietic stem cell transplantation and should be a criterion for donor selection.

Donor Lymphocyte Infusion in Hematologic Malignancies—Good to be Fresh?

BACKGROUND Donor lymphocyte infusion (DLI) has been used with variable success in a variety of hematologic malignancies. PATIENTS AND METHODS We conducted a retrospective analysis of all patients who were treated with DLI for persistent or relapsed disease at the Temple University Bone Marrow Transplant Unit from July 1, 1993 to December 31, 2013 to evaluate the effect of the type of DLI (fresh vs. cryopreserved) on event-free survival (EFS) and overall survival (OS). Median follow-up was 64.8 months (range, 0.3-142.6 months). RESULTS We found that EFS and OS were similar between patients receiving cryopreserved cells and those receiving fresh DLI (median OS for cryopreserved cells, 0.39 years; median OS for fresh cells, 0.32 years; P = .793; median EFS for cryopreserved cells, 0.410 years; median EFS for fresh cells, 0.420 years; P = .4264). In the setting of relapsed disease, treatment with any chemotherapy regimen before receiving DLI did not significantly impact OS (n = 63; P = .2203) or EFS (n = 40; P = .542). A subgroup analysis limited to patients with acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS) (32 patients) showed that differences in OS and EFS between cryopreserved and fresh DLI approached significance (median OS for cryopreserved cells, 0.34 years; median OS for fresh cells, 0.17 years; P = .16; median EFS for cryopreserved cells, 0.37 years; median EFS for fresh cells, 0.094 years; P = 0.11). CONCLUSION We conclude that the use of fresh cells versus cryopreserved cells does not have an impact on outcomes, and selected patients can achieve long-term survival with DLI for treatment of relapse after transplantation, although the overall outcomes remain dismal.

Oral vancomycin prophylaxis is highly effective in preventing Clostridium difficile infection in allogeneic hematopoietic cell transplant recipients

BACKGROUND Clostridium difficile infection (CDI) is a leading cause of infectious complications in allogeneic hematopoietic cell transplant recipients (alloHCT). We sought to evaluate whether prophylactic oral vancomycin reduces the incidence of CDI in alloHCT recipients. METHODS We conducted a retrospective cohort study to examine the effectiveness of CDI prophylaxis with oral vancomycin, as compared to no prophylaxis, in 145 consecutive adult alloHCT recipients at the University of Pennsylvania between April 2015 and November 2016. Patients received oral vancomycin 125 mg twice daily, starting on admission and continuing until discharge. The primary outcome of interest was the association between oral vancomycin prophylaxis and CDI diagnosis. Secondary outcomes included graft-versus-host disease (GVHD) and relapse. RESULTS There were no cases of CDI in patients that received prophylaxis (0/90, 0%), whereas 11/55 (20%) patients who did not receive prophylaxis developed CDI (P < .001). Oral vancomycin prophylaxis was not associated with a higher risk of acute, grades 2-4 GVHD (subhazard ratio [sHR] 1.59; 95% confidence interval [CI] 0.88-2.89; P = .12), acute, grades 3-4 GVHD (sHR 0.65; 95% CI 0.25-1.66; P = .36), or acute, grades 2-4 gastrointestinal GVHD (sHR 1.95; 95% CI 0.93-4.07; P = .08) at day 180 post-transplant. No associations between oral vancomycin and relapse or survival were observed. CONCLUSIONS Prophylaxis with oral vancomycin is highly effective in preventing CDI in alloHCT recipients without increasing the risk of graft-versus-host disease or disease relapse. Further evaluation via a prospective study is warranted.

Tocilizumab for the treatment of severe steroid-refractory acute graft-versus-host disease of the lower gastrointestinal tract

Steroid-refractory (SR) acute gastrointestinal (GI) graft-versus-host disease (GVHD) is associated with significant mortality in allogeneic hematopoietic cell transplantation recipients. We retrospectively evaluated the efficacy of tocilizumab for the treatment of SR biopsy-proven acute lower GI GVHD in 16 consecutive adult transplant recipients between October 2015 and July 2016. Tocilizumab 8 mg/kg was administered every 2 weeks until achievement of complete response, defined as resolution of all manifestations of GI GVHD, or until patients had progression or initiation of other therapy. Ten of 16 patients (62.5%; 95% CI, 0.39–82) achieved a complete response after a median time of 11 days (range, 2–28 days) from tocilizumab initiation. The median time to response onset (improvement in stage by at least 1) was 1 day (range, 1–4 days). Tocilizumab was administered at a median of 9 days (range, 3–75 days) from GVHD diagnosis and 10 days (range, 3–75 days) from initiation of high-dose steroids. At a median follow-up of 7.6 months (range, 0.8–27.7 months) from initiation of tocilizumab, 6/16 (37.5%) patients are alive and free of their underlying hematologic malignancy. Tocilizumab appears to be a highly active agent for the treatment of severe SR lower GI acute GVHD.