Craig E. Eckfeldt

The molecular repertoire of the 'almighty' stem cell

Stem cells share the defining characteristics of self-renewal, which maintains or expands the stem-cell pool, and multi-lineage differentiation, which generates and regenerates tissues. Stem-cell self-renewal and differentiation are influenced by the convergence of intrinsic cellular signals and extrinsic microenvironmental cues from the surrounding stem-cell niche, but the specific signals involved are poorly understood. Recently, several studies have sought to identify the genetic mechanisms that underlie the stem-cell phenotype. Such a molecular road map of stem-cell function should lead to an understanding of the true potential of stem cells.

Functional analysis of human hematopoietic stem cell gene expression using zebrafish.

Although several reports have characterized the hematopoietic stem cell (HSC) transcriptome, the roles of HSC-specific genes in hematopoiesis remain elusive. To identify candidate regulators of HSC fate decisions, we compared the transcriptome of human umbilical cord blood and bone marrow CD34+CD33−CD38−Rholoc-kit+ cells, enriched for hematopoietic stem/progenitor cells with CD34+CD33−CD38−Rhohi cells, enriched in committed progenitors. We identified 277 differentially expressed transcripts conserved in these ontogenically distinct cell sources. We next performed a morpholino antisense oligonucleotide (MO)-based functional screen in zebrafish to determine the hematopoietic function of 61 genes that had no previously known function in HSC biology and for which a likely zebrafish ortholog could be identified. MO knock down of 14/61 (23%) of the differentially expressed transcripts resulted in hematopoietic defects in developing zebrafish embryos, as demonstrated by altered levels of circulating blood cells at 30 and 48 h postfertilization and subsequently confirmed by quantitative RT-PCR for erythroid-specific hbae1 and myeloid-specific lcp1 transcripts. Recapitulating the knockdown phenotype using a second MO of independent sequence, absence of the phenotype using a mismatched MO sequence, and rescue of the phenotype by cDNA-based overexpression of the targeted transcript for zebrafish spry4 confirmed the specificity of MO targeting in this system. Further characterization of the spry4-deficient zebrafish embryos demonstrated that hematopoietic defects were not due to more widespread defects in the mesodermal development, and therefore represented primary defects in HSC specification, proliferation, and/or differentiation. Overall, this high-throughput screen for the functional validation of differentially expressed genes using a zebrafish model of hematopoiesis represents a major step toward obtaining meaningful information from global gene profiling of HSCs.

Genome-Wide Reverse Genetics Framework to Identify Novel Functions of the Vertebrate Secretome

Background Understanding the functional role(s) of the more than 20,000 proteins of the vertebrate genome is a major next step in the post-genome era. The approximately 4,000 co-translationally translocated (CTT) proteins – representing the vertebrate secretome – are important for such vertebrate-critical processes as organogenesis. However, the role(s) for most of these genes is currently unknown. Results We identified 585 putative full-length zebrafish CTT proteins using cross-species genomic and EST-based comparative sequence analyses. We further investigated 150 of these genes (Figure 1) for unique function using morpholino-based analysis in zebrafish embryos. 12% of the CTT protein-deficient embryos resulted in specific developmental defects, a notably higher rate of gene function annotation than the 2%–3% estimate from random gene mutagenesis studies. Conclusion(s) This initial collection includes novel genes required for the development of vascular, hematopoietic, pigmentation, and craniofacial tissues, as well as lipid metabolism, and organogenesis. This study provides a framework utilizing zebrafish for the systematic assignment of biological function in a vertebrate genome.

Microenvironmental influences on human B-cell development

Summary: Mammalian B‐cell development can be viewed as a developmental performance with several acts. The acts are represented by checkpoints centered around commitment to the B‐lineage and functional Ig gene rearrangement – culminating in expression of the pre‐B‐cell receptor (pre‐BCR) and the BCR. Progression of cells through these checkpoints is profoundly influenced by the fetal liver and adult bone marrow (BM) stromal cell microenvironments. Our laboratory has developed a model of human B‐cell development that utilizes freshly isolated/non‐transformed human BM stromal cells as an in vitro microenvironment. Human CD34+ hematopoietic stem cells plated in this human BM stromal cell microenvironment commit to the B lineage and progress through the pre‐BCR and BCR checkpoints. This human BM stromal cell microenvironment also provides survival signals that prevent apoptosis in human B‐lineage cells. Human B‐lineage cells exhibit differential expression of Notch receptors and human BM stromal cells express the Notch ligand Jagged‐1. These results suggest a potential role for Notch in regulating B‐lineage commitment and/or progression through the pre‐BCR and BCR checkpoints.

Notch-1 and Notch-2 exhibit unique patterns of expression in human B-lineage cells

The Notch genes encode a conserved family of receptors that influence developmental fate in many species. Prior studies have indicated that Notch-1 and Notch-2 signaling influence the development of hematopoietic stems cells and thymocytes, but little is known regarding Notch expression and function in B-lineage cells. We analyzed the expression of Notch receptors and Notch ligands in human B-lineage cells and bone marrow (BM) stromal cells. Notch-1 mRNA and protein is expressed throughout normal B cell development and in leukemic B-lineage cells. In contrast, Notch-2 expression is limited to pre-B cells expressing low levels of surface μ. The Notch ligand Delta is expressed in BM B-lineage cells. The Notch ligand Jagged-1 is not expressed in B-lineage cells, but is expressed in BM stromal cells. These results suggest a model wherein lateral signaling between Notch and Delta on B-lineage cells and/or Notch/Jagged-1 interactions between B-lineage cells and BM stromal cells may regulate human B cell development.

Stat5 is critical for the development and maintenance of myeloproliferative neoplasm initiated by Nf1 deficiency

Juvenile myelomonocytic leukemia is a rare myeloproliferative neoplasm characterized by hyperactive RAS signaling. Neurofibromin1 (encoded by the NF1 gene) is a negative regulator of RAS activation. Patients with neurofibromatosis type 1 harbor loss-of-function mutations in NF1 and have a 200- to 500-fold increased risk of juvenile myelomonocytic leukemia. Leukemia cells from patients with juvenile myelomonocytic leukemia display hypersensitivity to certain cytokines, such as granulocyte-macrophage colony-stimulating factor. The granulocyte-macrophage colony-stimulating factor receptor utilizes pre-associated JAK2 to initiate signals after ligand binding. JAK2 subsequently activates STAT5, among other downstream effectors. Although STAT5 is gaining recognition as an important mediator of growth factor signaling in myeloid leukemias, the contribution of STAT5 to the development of hyperactive RAS-initiated myeloproliferative disease has not been well described. In this study, we investigated the consequence of STAT5 attenuation via genetic and pharmacological approaches in Nf1-deficient murine models of juvenile myelomonocytic leukemia. We found that homozygous Stat5 deficiency extended the lifespan of Nf1-deficient mice and eliminated the development of myeloproliferative neoplasm associated with Nf1 gene loss. Likewise, we found that JAK inhibition with ruxolitinib attenuated myeloproliferative neoplasm in Nf1-deficient mice. Finally, we found that primary cells from a patient with KRAS-mutant juvenile myelomonocytic leukemia displayed reduced colony formation in response to JAK2 inhibition. Our findings establish a central role for STAT5 activation in the pathogenesis of juvenile myelomonocytic leukemia and suggest that targeting this pathway may be of clinical utility in these patients.

Allogeneic Hematopoietic Cell Transplantation for Older Patients: Prognosis Determined by Disease Risk Index

The treatment of elderly patients with advanced hematological malignancies has expanded to include reduced-intensity conditioning (RIC) allogeneic hematopoietic cell transplantation (alloHCT) as a potentially curative option. We studied the association between Disease Risk Index (DRI) and clinical outcomes of 196 elderly patients (median age, 64.8; range, 60 to 75 years) with hematological malignancies receiving RIC alloHCT (2000 to 2014). Donors were related and unrelated adults (n = 100, 51.1%) or umbilical cord blood (n = 96, 48.9%). DRI classified 12 patients (6.1%) as low risk (LR), 146 patients (74.5%) as intermediate risk (IR), and 38 patients (19.4%) as high risk (HR). Two-year overall survival (OS) was 47% (52% for LR/IR versus 29% for HR, P < .01) and 2-year disease-free survival was 39% (44% for LR/IR versus 21% for HR, P < .01). Relapse incidence was 30% (26% for LR/IR versus 44% for HR, P < .01). Treatment-related mortality was 29% at 2 years; this was similar for all DRI groups. In multiple regression analysis, HR DRI was associated with increased risk of relapse (hazard ratio, 2.07; 95% confidence interval [CI], 1.34 to 3.33; P = .02) and treatment failure (hazard ratio, 2.07; 95% CI, 1.35 to 3.18; P < .01) and decreased OS (hazard ratio, 2.11; 95% CI, 1.34 to 3.33; P < .01). In elderly patients, DRI is a significant prognostic factor for post-transplantation relapse, treatment failure, and mortality. Because of increased risk of relapse leading to poor survival in HR DRI, participation in clinical trials offering relapse prevention strategies after RIC alloHCT should be encouraged when available.

Umbilical cord blood transplantation is a suitable option for consolidation of acute myeloid leukemia with FLT3-ITD

Internal tandem duplication (ITD) of the FMS-like tyrosine kinase (FLT3) gene (FLT3-ITD) is present in 10%–30% of AML.1 FLT3-ITD+ AML is associated with an increased risk of relapse and shorter overall survival.1,2 Because of this, allogeneic hematopoietic cell transplantation (alloHCT) has been commonly used for consolidation of FLT3-ITD+ AML patients. Although direct comparisons are limited, outcomes after alloHCT generally compare favorably to chemotherapy-based approaches with overall survival (OS) at two or more years as high as 60%, although results are variable.2–5 Unfortunately, many patients lack a suitable HLA-matched donor, precluding alloHCT consolidation. Umbilical cord blood (UCB) transplantation provides an alternative donor source for alloHCT and has given promising results in many hematologic malignancies.6–10 The delayed immune reconstitution, functional T-cell recovery, and lower incidence of graft-versus-host-disease (GvHD) following UCB transplantation raises concerns about a compromised graft-versus-leukemia effect and increased risk of disease relapse;11 however, this has not translated into inferior outcomes for UCB transplantation in general.12,13 How UCB transplantation performs for specific high-risk AML subsets, such as FLT3-ITD+ AML, is not known. To address this, we analyzed the outcomes of UCB transplantation for FLT3-ITD+ AML at our center. We prospectively analyzed data collected from AML patients undergoing first alloHCT between 2008 and 2014 using the University of Minnesota Blood and Marrow Transplantation Database. Patients gave their consent and were treated according to protocols approved by our Institutional Review Board and registered at clinicaltrials.gov. Data on pre-transplantation comorbidities were collected using the HCT-specific comorbidity index (HCT-CI)14 and were categorized as low-risk (score 0), intermediate-risk (score 1–2), or high-risk (score ≥3). Cytogenetic data (G-banded karyotype and/or FISH analyses) at diagnosis were classified according to the Southwest Oncology Group (SWOG) cytogenetic risk classification system.15 FLT3 mutation status was analyzed using DNA from blood or bone marrow using multiplex polymerase chain reaction (PCR), as previously described.16 The PCR products were analyzed by capillary electrophoresis on an ABI 3130 (Foster City, CA, USA) before and after restriction enzyme digest. The FLT3-ITD mutation was identified by the presence of a peak size greater than the 330 base pair wild-type PCR product. Leukemia-free survival (LFS) and complete remission (CR) were defined according to the International Working Group criteria.17 All patients were in CR at the time of alloHCT, as determined by a bone marrow biopsy performed within less than four weeks before alloHCT. The presence of minimal residual disease (MRD) at the time of transplantation was determined by flow cytometry, cytogenetic (FISH/G-banding), and FLT3-ITD mutation testing in some patients. UCB grafts were matched at 4–6 of 6 HLA-A, -B (antigen level) and -DRB1 (allele level) loci to the recipient, and, in patients receiving two UCB units, were matched to each other. UCB units were required to have dose of at least 2.0×107 total nucleated cells (TNC)/kg with a target cell dose at least 3.0×107 TNC/kg. Reduced intensity conditioning (RIC) regimens included cyclophosphamide (50 mg/kg IV on day −6), fludarabine (30–40 mg/m2 IV daily from days −6 through −2) and total body irradiation (TBI) (200 cGy on day −1) or fludarabine (30 mg/m2 IV daily from days −6 through −2) and busulfan (3.2 mg/kg IV daily on days −5 and −4). Myeloablative conditioning (MAC) regimens included cyclophosphamide (60 mg/kg intravenously daily for 2 days) and 1320 cGy TBI in 8 fractions. Equine anti-thymocyte globulin (ATG, 15 mg/kg) was administered every 12 hours for six doses for patients who had not received chemotherapy within three months of transplantation. GvHD prophylaxis consisted of cyclosporine A (CSA) and mycophenolate mofetil (MMF) or sirolimus plus MMF. MMF was discontinued on day +30. Granulocyte-colony stimulating factor (G-CSF) was administered to all patients from day +1 until the absolute neutrophil count was more than 2.5×109/L for two days. Patients received institutional standard antimicrobial prophylaxis with fungal, bacterial, and viral directed antibiotics. One FLT3-ITD+ AML patient received a tyrosine kinase inhibitor (TKI) before alloHCT for remission induction. No patient received TKI maintenance therapy after alloHCT. The Kaplan-Meier method was used to estimate overall and disease-free survival from day of HCT. The cumulative incidence function with competing risks was used to estimate cumulative incidence of relapse (CIR) and non-relapse mortality (NRM). Statistics were calculated using R software v.3.0.2. Patients’ characteristics are summarized in Table 1. UCB transplantation was performed for 22 FLT3-ITD+ and 44 FLT3-ITD− AML patients. The FLT3-ITD+ and FLT3-ITD− groups were well matched for sex, disease status, HLA-matching, GvHD prophylaxis regimen, performance status, and comorbidities. The FLT3-ITD+ group had higher white blood cell (WBC) counts at diagnosis and more intermediate and fewer unfavorable risk cytogenetics than FLT3-ITD− patients. FLT3-ITD+ patients were younger than FLT3-ITD− patients and were more likely to receive a myeloablative conditioning regimen. The FLT3-ITD mutation was not detected in any of the 15 FLT3-ITD+ patients who were tested at the time of transplantation. Flow cytometric evidence of MRD seemed more common in the FLT3-ITD+ group. Two of the 5 FLT3-ITD+ patients with flow cytometric evidence of MRD at transplant tested negative for FLT3-ITD by molecular testing. Table 1. Patients’, disease and donors’ characteristics. Our analysis revealed that the 2-year CIR was similar in the FLT3-ITD+ (29%, 95%CI: 8%–50%), FLT3-ITD− (36%, 95%CI: 20%–51%) (Figure 1A). NRM was 23% (95%CI: 4%–41%) and 18% (95%CI: 6%–30%) for the FLT3-ITD+ and FLT3-ITD− groups at one year, respectively. Two-year LFS years was similar for FLT3-ITD+ (48%, 95%CI: 31%–7%) and FLT3-ITD− (37%, 95%CI: 25%–56%) groups (Figure 1B). OS at two years was also similar for the FLT3-ITD+ (47%, 95%CI: 29–75%) and FLT3-ITD− (42%, 95%CI: 29%–61%) groups (Figure 1C). OS at two years for patients with MRD+ (n=9) and MRD− (n=54) by flow cytometry was 13% (95%CI: 1–43, n=9) and 49% (95%CI: 35–62), respectively. Figure 1. Two-year (A) cumulative incidence of relapse (CIR), (B) leukemia-free survival (LFS) and (C) overall survival (OS) for FLT3-ITD+ and FLT3-ITD− AML cohorts. This is the first study providing results of UCB transplantation specifically for FLT3-ITD+ AML patients. The 2-year CIR of 29% and OS of 47% for UCB transplantation for FLT3-ITD+ AML in our series were similar to that of FLT3-ITD− patients. Moreover, our results are similar to published outcomes following transplantation for FLT3-ITD+ AML using HLA-matched sibling, HLA-haploidentical, or HLA-matched unrelated donors and superior to the 20%–30% survival that has been reported with chemotherapy consolidation alone.3,4,18,19 Therefore, UCB transplantation seems to overcome the adverse effects of FLT3-ITD+ on AML. This is consistent with standard/conventional donor (sibling or unrelated donor) alloHCT studies that have demonstrated not only lower rates of disease relapse,18 but also improved OS for FLT3-ITD+ AML who undergo alloHCT, particularly patients with a higher FLT3-ITD to wild-type FLT3 allelic ratio.3,4,19 The patient population and transplantation characteristics were homogenous, data were detailed [e.g. tyrosine kinase inhibitor (TKI) use] FLT3-ITD and the size of the study was reasonable (given frequency of FLT-ITD3 mutations and UCB transplantation) for a single center study. Moreover, the encouraging results seen for FLT3-ITD+ AML patients in this analysis are consistent with our previous experience with UCB transplantation for a variety of high-risk hematologic malignancies.6–9 While very few patients in our cohort received FLT3-targeted TKI therapy, the role of TKI therapy before and/or after transplantation is a topic of intense interest that warrants further investigation.20,21 Using FLT3 mutation as an MRD marker has not been standardized or demonstrated in large studies; however, several small studies have suggested that FLT3 mutations may be useful as an MRD marker, especially when sensitive methods are employed.22–25 In our limited numbers, FLT3-ITD mutations was negative in all tested patients while some of them had residual disease by other methods, possibly due to loss of the FLT3-ITD. Change in FLT3 mutations status at relapse (including either gain or loss of mutation) is a well-known phenomenon that occurs in a relatively small subset of patients. FLT3 mutation testing will not be useful as an MRD marker in patients who lose their FLT3 mutation; however, these patients have better OS and a longer time to relapse than patients who gain or retain FLT3 mutations.26,27 In line with reported studies by us and others,28–30 the presence of MRD by flow cytometry might be associated with a lower OS after alloHCT regardless of FLT3-ITD mutation status; however, considerable caution should be exercised given that only a very small number of patients had MRD positivity. Overall, our findings support the view that UCB is a safe and effective donor source for transplantation of FLT3-ITD+ AML, thereby expanding the donor options for these high-risk patients. This is particularly important for FLT3-ITD+ AML patients who have no suitable sibling donor and who, during the search for an unrelated donor, will probably experience rapid and frequent relapses that would preclude alloHCT.

Ras oncogene-independent activation of RALB signaling is a targetable mechanism of escape from NRAS(V12) oncogene addiction in acute myeloid leukemia

Somatic mutations that lead to constitutive activation of NRAS and KRAS proto-oncogenes are among the most common in human cancer and frequently occur in acute myeloid leukemia (AML). An inducible NRAS(V12)-driven AML mouse model has established a critical role for continued NRAS(V12) expression in leukemia maintenance. In this model genetic suppression of NRAS(V12) expression results in rapid leukemia remission, but some mice undergo spontaneous relapse with NRAS(V12)-independent (NRI) AMLs providing an opportunity to identify mechanisms that bypass the requirement for Ras oncogene activity and drive leukemia relapse. We found that relapsed NRI AMLs are devoid of NRAS(V12) expression and signaling through the major oncogenic Ras effector pathways, phosphatidylinositol-3-kinase and mitogen-activated protein kinase, but express higher levels of an alternate Ras effector, Ralb, and exhibit NRI phosphorylation of the RALB effector TBK1, implicating RALB signaling in AML relapse. Functional studies confirmed that inhibiting CDK5-mediated RALB activation with a clinically relevant experimental drug, dinaciclib, led to potent RALB-dependent antileukemic effects in human AML cell lines, induced apoptosis in patient-derived AML samples in vitro and led to a 2-log reduction in the leukemic burden in patient-derived xenograft mice. Furthermore, dinaciclib potently suppressed the clonogenic potential of relapsed NRI AMLs in vitro and prevented the development of relapsed AML in vivo. Our findings demonstrate that Ras oncogene-independent activation of RALB signaling is a therapeutically targetable mechanism of escape from NRAS oncogene addiction in AML.

RALB provides critical survival signals downstream of Ras in acute myeloid leukemia.

Mutations that activate RAS proto-oncogenes and their effectors are common in acute myeloid leukemia (AML); however, efforts to therapeutically target Ras or its effectors have been unsuccessful, and have been hampered by an incomplete understanding of which effectors are required for AML proliferation and survival. We investigated the role of Ras effector pathways in AML using murine and human AML models. Whereas genetic disruption of NRAS(V12) expression in an NRAS(V12) and Mll-AF9-driven murine AML induced apoptosis of leukemic cells, inhibition of phosphatidylinositol-3-kinase (PI3K) and/or mitogen-activated protein kinase (MAPK) signaling did not reproduce this effect. Conversely, genetic disruption of RALB signaling induced AML cell death and phenocopied the effects of suppressing oncogenic Ras directly – uncovering a novel role for RALB signaling in AML survival. Knockdown of RALB led to decreased phosphorylation of TBK1 and reduced BCL2 expression, providing mechanistic insight into RALB survival signaling in AML. Notably, we found that patient-derived AML blasts have higher levels of RALB-TBK1 signaling compared to normal blood leukocytes, supporting a pathophysiologic role for RALB signaling for AML patients. Overall, our work provides new insight into the specific roles of Ras effector pathways in AML and has identified RALB signaling as a key survival pathway.