Patrick J. Kerzic

Prospective study of 174 de novo acute myelogenous leukemias according to the WHO classification: subtypes, cytogenetic features and FLT3 mutations

Abstract:  We report a prospective study of 174 unselected adult de novo acute myeloid leukemia (AML) cases diagnosed using the WHO classification. Of those, 57 (33%) were AML with recurrent cytogenetic abnormalities, 41 were (24%) AML with multilineage dysplasia, 74 (42%) were AML not otherwise categorized, and two were acute leukemias of ambiguous lineage. Clonal cytogenetic abnormalities were detected in 64% of the WHO AML cases with t(15;17) (15%), t(8;21) (12%), +8 (11%), −7/del7q (8%) and del9q (5%) being the most common ones. The FLT3/ITD mutations (FMS‐like tyrosine kinase 3/internal tandem duplication) were observed in 12% of the WHO AML cases, which is much lower than ones in the literature, while the 6% incidence of the FLT3‐activating loop mutations (either FLT3/D835 or FLT3/I836) was comparable with others. Both mutations were associated with leukocytosis. Our study also suggests that the FLT3 mutations are biomarkers independent of cytogenetic characteristics.

Inhibition of NF-κB by hydroquinone sensitizes human bone marrow progenitor cells to TNF-α-induced apoptosis

Suppression of hematopoiesis is an important mechanism governing blood cell formation. Factors such as tumor necrosis factor alpha (TNF-alpha) inhibit proliferation and colony-forming activity of bone marrow cells and activate nuclear factor kappa B (NF-kappaB) in multiple cell types. Activated NF-kappaB is required for many cells to escape apoptosis, including hematopoietic progenitor cells (HPC). The benzene metabolite hydroquinone (HQ) alters cytokine response and induces cell death in HPC, and inhibits NF-kappaB activation in T and B cells. Therefore, we studied the potential role of HQ-induced NF-kappaB inhibition in a hematopoietic cell line (TF-1) and primary HPC in rendering these cells susceptible to TNF-alpha-induced apoptosis. We demonstrate in both cell types that TNF-alpha activates NF-kappaB, and HQ exposure inhibits activation of NF-kappaB by TNF-alpha in a dose dependent manner. We further investigated the ability of HQ to potentiate TNF-alpha-induced apoptosis in these cells, and found that HQ sensitized the cells to the pro-apoptotic effect of TNF-alpha. These results suggest that NF-kappaB plays a key role in HPC survival, and that HQ-induced inhibition of NF-kappaB leaves these cells susceptible to cytokine-induced apoptosis. These effects may play a role in the suppression of hematopoiesis seen in some benzene exposed individuals.

Acute myeloid leukemia following exposure to benzene more closely resembles de novo than therapy related-disease.

Benzene (Bz) is widely regarded as a prototype environmental leukemogen and individuals chronically exposed are at risk for myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). It is widely assumed that initiation and pathogenesis of AML following Bz exposure (Bz‐AML) is similar or identical to therapy‐related AML (t‐AML), in which clonal cytogenetic abnormalities, including aneuploidy, are initiating events. However, this assumption is not supported by studies reporting actual disease outcomes together with cytogenetic analyses. Here, using clinically relevant cytogenetic, hematologic, and epidemiological methods, we directly show for 722 consecutive AML cases that the pattern of clonal cytogenetic abnormalities encountered in Bz‐exposed cases (n = 78) more closely resembles de novo‐AML than t‐AML. The prevalence of aneuploidy in Bz‐exposed‐ and de novo‐AML cases was identical (23%), and no significant increases in −5/5q− (RR = 0.79) (95% CI: 0.29–2.12) or −7/7q− (RR = 1.27) (95% CI: 0.55–2.92) abnormalities were observed between Bz‐ vs de novo‐AML, respectively. Previous studies have suggested a role for autoimmunity in Bz related MDS including immune mediated inflammatory features and positive responses to immunosuppressive therapy which are indistinguishable from those reported in MDS with low risk of progression to AML. These observations are more consistent with an epigenetic model for initiation of Bz‐AML in which altered homeostatic regulation in the bone marrow niche, not direct cytogenetic injury, predominates in the initial development of the leukemic stem cell phenotype, a mechanism biologically distinct from previous models of clonal cytogenetic injury. These findings are important for further understanding the biological basis of AML, particularly in environmental and occupational settings.

PU.1 phosphorylation correlates with hydroquinone-induced alterations in myeloid differentiation and cytokine-dependent clonogenic response in human CD34(+) hematopoietic progenitor cells.

The transcriptional regulatory factor PU.1 is important for the regulation of a diverse group of hematopoietic and myeloid genes. Posttranslational phosphorylation of PU.1 has been demonstrated in the regulation of a variety of promoters in normal cells. In leukemia cells, differing patterns of PU.1 phosphorylation have been described among acute myelogenous leukemia (AML) subtypes. Therefore, we hypothesized that modulation of PU.1-dependent gene expression might be a molecular mediator of alterations in myeloid cell growth and differentiation that have been demonstrated to be early events in benzene-induced leukemogenesis. We found that freshly isolated human CD34+ hematopoietic progenitor cells (HPC) exhibit multiple PU.1-DNA binding species that represent PU.1 proteins in varying degrees of phosphorylation states as determined by phosphatase treatment in combination with electrophoretic mobility shift assay (EMSA). Maturation of granulocyte and monocyte lineages is also accompanied by distinct changes in PU.1-DNA binding patterns. Experiments reveal that increasing doses of the benzene metabolite, hydroquinone (HQ) induce a time-and dose-dependent alteration in the pattern of PU.1-DNA binding in cultured human CD34+ cells, corresponding to hyperphosphorylation of the PU.1 protein. HQ-induced alterations in PU.1-DNA binding are concomitant with a sustained immature CD34+ phenotype and cytokine-dependent enhanced clonogenic activity in cultured human HPC. These results suggest that HQ induces a dysregulation in the external signals modulating PU.1 protein phosphorylation and this dysregulation may be an early event in the generation of benzene-induced AML.

Cytogenetics in benzene-associated myelodysplastic syndromes and acute myeloid leukemia: new insights into a disease continuum.

Hematopoiesis in health and disease results from complex interactions between primitive hematopoietic stem cells (HSCs) and the extrinsic influences of other cells in the bone marrow (BM) niche. Advances in stem cell biology, molecular genetics, and computational biology reveal that the immortality, self‐renewal, and maintenance of blood homeostasis generally attributed to individual HSCs are functions of the cells’ behavior in the normal BM environment. Here we discuss how these advances, together with results of outcomes‐based clinical epidemiology studies, provide new insight into the importance of epigenetic events in leukemogenesis. For the chemical benzene (Bz), development of myeloid neoplasms depends predominantly on alterations within the microenvironments in which they arise. The primary persistent disease in Bz myelotoxicity is myelodysplastic syndrome, which precedes cytogenetic injury. Evidence indicates that acute myeloid leukemia arises as a secondary event, subsequent to evolution of the leukemia‐initiating cell phenotype within the altered BM microenvironment. Further explorations into the nature of chemical versus de novo disease should consider this mechanism, which is biologically distinct from previous models of clonal cytogenetic injury. Understanding alterations of homeostatic regulation in the BM niche is important for validation of models of leukemogenesis, monitoring at‐risk populations, and development of novel treatment and prevention strategies.

Mutant forms of bcr-abl deficient in inducing abi degradation show different leukemogenic activity

Abstract Bcr-Abl elicits the ubiquitin-dependent degradation of Abl interactor (Abi) proteins, a family of proteins that antagonize the oncogenic potential of Abl. Significantly, expression of the Abi proteins is lost in cell lines and bone marrow cells isolated from patients with aggressive Bcr-Abl-positive leukemias. To determine the role of Abi degradation in Bcr-Abl-induced leukemogenesis, we have mapped the sequences in Bcr-Abl that are required for inducing Abi degradation. The deletion of C-terminal proline-rich sequences (p185 Bcr-AblΔ924-1018 ) severely impairs Bcr-Abl-induced Abi degradation. The double deletions of the SH3 domain and C-terminal proline-rich sequences (p185 Bcr-AblΔ924-1018 ) completely abolish the degradation of Abi proteins. We then compared the leukemogenic activity of these mutant forms of Bcr-Abl to that of the wild type Bcr-Abl in a murine bone marrow transduction/transplantation model. Like wild type P185 Bcr-Abl , both p185 Bcr-AblΔ924-1018 and p185 Bcr-AblΔSH3Δ924-1018 transform mouse bone marrow cells with similar potency, as judged by factor-independent growth in an agar colony assay. The mutant forms of Bcr-Abl, however, showed different leukemogenic activity in mice. Mice reconstituted with wild type P185 Bcr-Abl transduced marrow cells developed chronic myeloid leukemia (CML)-like disease and died in 5–7 weeks after transplantation. The peripheral white blood cell (WBC) count of these mice was significantly elevated and the mice had enlarged spleens. Mice reconstituted with p185 Bcr-AblΔ918-1018 transduced marrow cells also died in 5–7 weeks. The majority of these mice, however, did not show the elevated WBC count or the spenomegaly, suggesting that they died of a disease other than CML. Furthermore, mice reconstituted with p185 Bcr-AblΔSH3Δ924-1018 transduced marrow cells survived much longer post-transplantation without the elevated WBC count. These results are consistent with the hypothesis that Abi degradation is involved in the development of Bcr-Abl positive CML. Studies are now in progress to define the pathology of diseases caused by mutant forms of Bcr-Abl.

Allogeneic cord blood recipients have delayed immune recovery associated with a deficiency in dendritic cells

Abstract Analysis of patients receiving ex-vivo expanded cord blood (ExCB) has demonstrated delayed recovery of immune cells. We therefore monitored T lymphocytes (CD3+, CD4+, and CD8+ cells) and blood dendritic cells defined as (CMRF44+, CD14−, and CD19− DCs) in this patient population. Cord blood products have low, and in some cases lack DCs. Prior to transplant, patients had DC levels of 2.03 × 10 4 /ml and CD3 levels of 7.58 × 10 5 /ml (n = 11). However, thirty days post-transplant, these ExCB recipients had a deficiency of DC (3.91 × 10 2 /ml) and CD3 cell levels (4.2 × 10 4 /ml) that were 18-fold lower than pre-transplant levels (n = 5). As a control for immunosuppressive therapy we analyzed matched unrelated peripheral blood progenitor cells (PBPC) and bone marrow recipients. Initial results show a return of CD3 cells to pre-transplant levels coincidental with the presence of DCs by day 30-post transplant. This suggests that the delayed recovery in ExCB recipients was not due to immune suppressive therapy. For a comparison of immune recovery in ExCB recipients, we have analyzed DC and T cell levels of patients receiving autologous PBPC. These patients had comparable pre-transplant levels of DCs and CD3 cells (1.72 × 10 3 /ml and 1.09 × 10 6 /ml, respectively n=24). However, thirty days after autologous PBPC transplant, the DCs and CD3 cells had returned to pre-transplant levels (8.26 × 10 3 /ml and 3.49 × 10 5 /ml, respectively n=9). Mouse models have demonstrated the requirement of DCs for peripheral proliferation of T cells. We propose that the delayed recovery to T cells in ExCB recipients is due to low levels or absence of DCs in CB products and the subsequent lack of DC in the blood of recipients post transplant.