Cdc42-Borg4-Septin7 axis regulates HSC polarity and function.

Aging of hematopoietic stem cells (HSCs) is caused by the elevated activity of the small RhoGTPase Cdc42 and an apolar distribution of proteins. Mechanisms by which Cdc42 activity controls polarity of HSCs are not known. Binder of RhoGTPases proteins (Borgs) are known effector proteins of Cdc42 that are able to regulate the cytoskeletal Septin network. Here, we show that Cdc42 interacts with Borg4, which in turn interacts with Septin7 to regulate the polar distribution of Cdc42, Borg4, and Septin7 within HSCs. Genetic deletion of either Borg4 or Septin7 results in a reduced frequency of HSCs polar for Cdc42 or Borg4 or Septin7, a reduced engraftment potential and decreased lymphoid-primed multipotent progenitor (LMPP) frequency in the bone marrow. Taken together, our data identify a Cdc42-Borg4-Septin7 axis essential for the maintenance of polarity within HSCs and for HSC function and provide a rationale for further investigating the role of Borgs and Septins in the regulation of compartmentalization within stem cells.

Aging of human hematopoietic stem cells is linked to changes in Cdc42 activity.

In this study, we characterize age-related phenotypes of human hematopoietic stem cells (HSC). We report increased frequencies of HSC, hematopoietic progenitor cells and lineage negative cells in the elderly but a decreased frequency of multi-lymphoid progenitors. Aged human HSC further exhibited a delay in initiating division ex vivo though without changes in their division kinetics. The activity of the small RhoGTPase Cdc42 was elevated in aged human hematopoietic cells and we identified a positive correlation between Cdc42 activity and the frequency of HSC upon aging. The frequency of human HSC polar for polarity proteins was, similar to the mouse, decreased upon aging, while inhibition of Cdc42 activity via the specific pharmacological inhibitor of Cdc42 activity, CASIN, resulted in re-polarization of aged human HSC with respect to Cdc42. Elevated activity of Cdc42 in aged HSC thus contributed to age-related changes in HSC. Xenotransplant, using NBSGW mice as recipients, showed elevated chimerism in recipients of aged compared to young HSC. Aged HSC treated with CASIN ex vivo displayed an engraftment profile similar to recipients of young HSC. Taken together, our work reveals strong evidence for a role of elevated Cdc42 activity in driving aging of human HSC, and similar to mice, this presents a likely possibility for attenuation of aging in human HSC.

Osteopontin attenuates aging-associated phenotypes of hematopoietic stem cells.

Upon aging, hematopoietic stem cells (HSCs) undergo changes in function and structure, including skewing to myeloid lineages, lower reconstitution potential and loss of protein polarity. While stem cell intrinsic mechanisms are known to contribute to HSC aging, little is known on whether age-related changes in the bone marrow niche regulate HSC aging. Upon aging, the expression of osteopontin (OPN) in the murine bone marrow stroma is reduced. Exposure of young HSCs to an OPN knockout niche results in a decrease in engraftment, an increase in long-term HSC frequency and loss of stem cell polarity. Exposure of aged HSCs to thrombin-cleaved OPN attenuates aging of old HSCs, resulting in increased engraftment, decreased HSC frequency, increased stem cell polarity and a restored balance of lymphoid and myeloid cells in peripheral blood. Thus, our data suggest a critical role for reduced stroma-derived OPN for HSC aging and identify thrombin-cleaved OPN as a novel niche informed therapeutic approach for ameliorating HSC phenotypes associated with aging.

Aged murine hematopoietic stem cells drive aging-associated immune remodeling.

Aging-associated remodeling of the immune system impairs its functional integrity and contributes to increased morbidity and mortality in the elderly. Aging of hematopoietic stem cells (HSCs), from which all cells of the adaptive immune system ultimately originate, might play a crucial role in the remodeling of the aged immune system. We recently reported that aging of HSCs is, in part, driven by elevated activity of the small RhoGTPase Cdc42 and that aged HSCs can be rejuvenated in vitro by inhibition of the elevated Cdc42 activity in aged HSCs with the pharmacological compound CASIN. To study the quality of immune systems stemming selectively from young or aged HSCs, we established a HSC transplantation model in T- and B-cell-deficient young RAG1-/- hosts. We report that both phenotypic and functional changes in the immune system on aging are primarily a consequence of changes in the function of HSCs on aging and, to a large extent, independent of the thymus, as young and aged HSCs reconstituted distinct T- and B-cell subsets in RAG1-/- hosts that mirrored young and aged immune systems. Importantly, aged HSCs treated with CASIN reestablished an immune system similar to that of young animals, and thus capable of mounting a strong immune response to vaccination. Our studies further imply that epigenetic signatures already imprinted in aged HSCs determine the transcriptional profile and function of HSC-derived T and B cells.

HPRT and Purine Salvaging Are Critical for Hematopoietic Stem Cell Function.

Adult hematopoietic stem cells (HSCs) maintain tissue homeostasis and regenerative capacity of the hematopoietic system through self-renewal and differentiation. Metabolism is recognized as an important regulatory entity controlling stem cells. As purine nucleotides are essential for metabolic functions, we analyzed the role of hypoxanthine guanine phosphoribosyl transferase (HPRT)-associated purine salvaging in HSCs. Here, we demonstrate that hematopoietic stem and progenitor cells (HSPCs) show a strong dependence on HPRT-associated purine salvaging. HSPCs with lower HPRT activity had a severely reduced competitive repopulation ability upon transplantation. Strikingly, HPRT deficiency resulted in altered cell-cycle progression, proliferation kinetics and mitochondrial membrane potential primarily in the HSC compartment, whereas more committed progenitors were less affected. Our data thus imply a unique and important role of HPRT and the purine salvage pathway for HSC function. Stem Cells 2019;37:1606-1614.

KDM6A, a histone demethylase, regulates stress hematopoiesis and early B-cell differentiation.

Histone methylases and demethylases regulate gene expression programs in hematopoiesis. The molecular function of the demethylase KDM6A in normal hematopoiesis and, in particular, for the hematopoietic stem and progenitor cell (HSPC) compartment remains only partially understood. Female but not male Kdm6a-/- HSPCs were functionally impaired in adoptive transfer experiments as well as upon proliferative stress induced by 5-fluorouracil. Loss of Kdm6a affected primarily early B cells and erythroid and myeloid progenitor cells with respect to both number and function. Global gene expression analyses revealed a shared altered gene signature in Kdm6a-/- pro-B and pre-B cells that is also present in HSPCs, supporting that altered B-cell differentiation in Kdm6a-/- animals is already initiated in HSPCs. Interestingly, loss of KDM6A did not affect the global level of methylation of H3K27, its presumed target, in hematopoietic cells. Our data indicate a critical role for KDM6A in the regulation of hematopoietic differentiation and differentiation-specific gene expression programs, with a prominent role in early B-cell differentiation that is likely independent of H3K27 methylation status.

The homeobox gene CDX2 is aberrantly expressed in most cases of acute myeloid leukemia and promotes leukemogenesis.

The homeobox transcription factor CDX2 plays an important role in embryonic development and regulates the proliferation and differentiation of intestinal epithelial cells in the adult. We have found that CDX2 is expressed in leukemic cells of 90% of patients with acute myeloid leukemia (AML) but not in hematopoietic stem and progenitor cells derived from normal individuals. Stable knockdown of CDX2 expression by RNA interference inhibited the proliferation of various human AML cell lines and strongly reduced their clonogenic potential in vitro. Primary murine hematopoietic progenitor cells transduced with Cdx2 acquired serial replating activity, were able to be continuously propagated in liquid culture, generated fully penetrant and transplantable AML in BM transplant recipients, and displayed dysregulated expression of Hox family members in vitro and in vivo. These results demonstrate that aberrant expression of the developmental regulatory gene CDX2 in the adult hematopoietic compartment is a frequent event in the pathogenesis of AML; suggest a role for CDX2 as part of a common effector pathway that promotes the proliferative capacity and self-renewal potential of myeloid progenitor cells; and support the hypothesis that CDX2 is responsible, in part, for the altered HOX gene expression that is observed in most cases of AML.

The Spindle Assembly Checkpoint Is Required for Hematopoietic Progenitor Cell Engraftment.

The spindle assembly checkpoint plays a pivotal role in preventing aneuploidy and transformation. Many studies demonstrate impairment of this checkpoint in cancer cells. While leukemia is frequently driven by transformed hematopoietic stem and progenitor cells (HSPCs), the biology of the spindle assembly checkpoint in such primary cells is not very well understood. Here, we reveal that the checkpoint is fully functional in murine progenitor cells and, to a lesser extent, in hematopoietic stem cells. We show that HSPCs arrest at prometaphase and induce p53-dependent apoptosis upon prolonged treatment with anti-mitotic drugs. Moreover, the checkpoint can be chemically and genetically abrogated, leading to premature exit from mitosis, subsequent enforced G1 arrest, and enhanced levels of chromosomal damage. We finally demonstrate that, upon checkpoint abrogation in HSPCs, hematopoiesis is impaired, manifested by loss of differentiation potential and engraftment ability, indicating a critical role of this checkpoint in HSPCs and hematopoiesis.

The prognostic value of MLL-AF9 detection in patients with t(9;11)(p22;q23)-positive acute myeloid leukemia.

BACKGROUND AND OBJECTIVES: Translocation (9;11) is the most common t(11q23) in acute myeloid leukemia (AML). A considerable number of patients with this cytogenetic abnormality relapse and die of their disease. We evaluated the clinical significance of minimal residual disease (MRD) monitoring in t(9;11)(p22;q23)-positive AML patients using real-time quantitative reverse transcriptase polymerase chain reaction (RQ-PCR) analysis. DESIGN AND METHODS: We identified 34 newly diagnosed patients with t(9;11)(p22;q23)-positive AML treated within three multicenter trials of the AML Study Group. MRD could be investigated by RQ-PCR in 19 patients during and after therapy. Because of the relatively low sensitivity of the RQ-PCR (10(-3) to 10(-4) at the cellular level), samples from RQ-PCR-negative patients were also analyzed by nested polymerase chain reaction (nPCR; sensitivity 10-4 to 10-5 at the cellular level). RESULTS: RQ-PCR monitoring revealed two groups of patients: group 1 (n=11) had negative RQ-PCR in all samples collected in hematologic complete remission whereas group 2 (n=8) had at least one positive RQ-PCR in samples collected in complete remission during therapy. Group 1 had a significantly lower cumulative incidence of relapse (p=0.004) and better overall survival (p=0.003) compared to group 2. nPCR did not add information to that gained from RQ-PCR. Molecular relapse was detected in two patients by RQ-PCR four and six weeks, respectively before hematologic relapse occurred. Quantitative MLL-AF9 levels at diagnosis or during and after therapy had no prognostic impact. INTERPRETATION AND CONCLUSIONS: Early achievement of sustained RQ-PCR negativity appears to be a prerequisite for long-term hematologic complete remission in t(9;11)-positive AML. Furthermore, RQ-PCR might be useful for early detection of relapse. Additional patients need to be studied to corroborate these findings.

Deregulated expression of EVI1 defines a poor prognostic subset of MLL-rearranged acute myeloid leukemias: a study of the German-Austrian Acute Myeloid Leukemia Study Group and the Dutch-Belgian-Swiss HOVON/SAKK Cooperative Group.

PURPOSE: To evaluate the prognostic value of ecotropic viral integration 1 gene (EVI1) overexpression in acute myeloid leukemia (AML) with MLL gene rearrangements. PATIENTS AND METHODS: We identified 286 patients with AML with t(11q23) enrolled onto German-Austrian Acute Myeloid Leukemia Study Group and Dutch-Belgian-Swiss Hemato-Oncology Cooperative Group prospective treatment trials. Material was available from 177 AML patients for EVI1 expression analysis. RESULTS: We divided 286 MLL-rearranged AMLs into three subgroups: t(9;11)(p22;q23) (44.8%), t(6;11)(q27;q23) (14.7%), and t(v;11q23) (40.5%). EVI1 overexpression (EVI1(+)) was found in 45.8% of all patients with t(11q23), with t(6;11) showing the highest frequency (83.9%), followed by t(9;11) at 40.0%, and t(v;11q23) at 34.8%. Concurrent gene mutations were rare or absent in all three subgroups. Within all t(11q23) AMLs, EVI1(+) was the sole prognostic factor, predicting for inferior overall survival (OS; hazard ratio [HR], 2.06; P = .003), relapse-free survival (HR, 2.28; P = .002), and event-free survival (HR, 1.79; P = .009). EVI1(+) AMLs with t(11q23) in first complete remission (CR) had a significantly better outcome after allogeneic transplantation compared with other consolidation therapies (5-year OS, 54.7% v 0%; Mantel-Byar, P = .0006). EVI1(-) t(9;11) AMLs had lower WBC counts, more commonly FAB M5 morphology, and frequently had additional trisomy 8 (39.6%; P < .001). Among t(9;11) AMLs, EVI1(+) again was the sole independent adverse prognostic factor for survival. CONCLUSION: Deregulated EVI1 expression defines poor prognostic subsets among AML with t(11q23) and AML with t(9;11)(p22;q23). Patients with EVI1(+) MLL-rearranged AML seem to benefit from allogeneic transplantation in first CR.

Monitoring of minimal residual disease in NPM1-mutated acute myeloid leukemia: a study from the German-Austrian acute myeloid leukemia study group.

PURPOSE: To evaluate the prognostic value of minimal residual disease (MRD) in patients with acute myeloid leukemia (AML) with NPM1 mutation (NPM1(mut)). PATIENTS AND METHOD: RNA-based real-time quantitative polymerase chain reaction (RQ-PCR) specific for the detection of six different NPM1(mut) types was applied to 1,682 samples (bone marrow, n = 1,272; blood, n = 410) serially obtained from 245 intensively treated younger adult patients who were 16 to 60 years old. RESULTS: NPM1(mut) transcript levels as a continuous variable were significantly associated with prognosis after each treatment cycle. Achievement of RQ-PCR negativity after double induction therapy identified patients with a low cumulative incidence of relapse (CIR; 6.5% after 4 years) compared with RQ-PCR-positive patients (53.0%; P < .001); this translated into significant differences in overall survival (90% v 51%, respectively; P = .001). After completion of therapy, CIR was 15.7% in RQ-PCR-negative patients compared with 66.5% in RQ-PCR-positive patients (P < .001). Multivariable analyses after double induction and after completion of consolidation therapy revealed higher NPM1(mut) transcript levels as a significant factor for a higher risk of relapse and death. Serial post-treatment assessment of MRD allowed early detection of relapse in patients exceeding more than 200 NPM1(mut)/10(4) ABL copies. CONCLUSION: We defined clinically relevant time points for NPM1(mut) MRD assessment that allow for the identification of patients with AML who are at high risk of relapse. Monitoring of NPM1(mut) transcript levels should be incorporated in future clinical trials to guide therapeutic decisions.

Prognostic impact of minimal residual disease in CBFB-MYH11-positive acute myeloid leukemia.

PURPOSE: To evaluate the prognostic impact of minimal residual disease (MRD) in patients with acute myeloid leukemia (AML) expressing the CBFB-MYH11 fusion transcript. PATIENTS AND METHODS: Quantitative reverse transcriptase polymerase chain reaction (PCR) was performed on 684 bone marrow (BM; n = 331) and/or peripheral blood (PB; n = 353) samples (median, 13 samples per patient) from 53 younger adult (16 to 60 years old) patients with AML treated in prospective German-Austrian AML Study Group treatment trials. Samples were obtained at diagnosis (BM, n = 45; PB, n = 48), during treatment course (BM, n = 153; PB, n = 122), and at follow-up (BM, n = 133; PB, n = 183). To evaluate the applicability of PB for MRD detection, 198 paired BM and PB samples obtained at identical time points were analyzed. RESULTS: The following three clinically relevant checkpoints were identified during consolidation and early follow-up that predicted relapse: achievement of PCR negativity in at least one BM sample during consolidation therapy (2-year relapse-free survival [RFS], 79% v 54% for PCR positivity; P = .035); achievement of PCR negativity in at least two BM or PB samples during consolidation therapy and early follow-up (< or = 3 months; 2-year RFS, P = .001; overall survival, P = .01); and conversion from PCR negativity to PCR positivity with copy ratios of more than 10 after consolidation therapy. Analysis of paired BM and PB samples revealed BM samples to be more sensitive during the course of therapy, whereas for follow-up, PB samples were equally informative. CONCLUSION: We defined clinically relevant MRD checkpoints that allow for the identification of patients with CBFB-MYH11-positive AML who are at high risk of relapse. Monitoring of CBFB-MYH11 transcript levels should be incorporated into future clinical trials to guide therapeutic decisions.

High EVI1 expression predicts outcome in younger adult patients with acute myeloid leukemia and is associated with distinct cytogenetic abnormalities.

PURPOSE The purpose of this study was to investigate frequency and prognostic significance of high EVI1 expression in acute myeloid leukemia (AML). PATIENTS AND METHODS A diagnostic assay detecting multiple EVI1 splice variants was developed to determine the relative EVI1 expression by single real-time quantitative polymerase chain reaction in 1,382 newly diagnosed adult patients with AML younger than 60 years. Patients were treated on four Dutch-Belgian HOVON (n = 458) and two German-Austrian AML Study Group protocols (n = 924). Results The EVI1 assay was tested in the HOVON cohort and validated in the AMLSG cohort. High EVI1 levels (EVI1(+)) were found with similar frequencies in both cohorts combined, with a 10.7% incidence (148 of 1,382). EVI1(+) independently predicted low complete remission (CR) rate (odds ratio, 0.54; P = .002), adverse relapse-free survival (RFS; hazard ratio [HR], 1.32; P = .05), and event-free survival (EFS; HR, 1.46; P < .001). This adverse prognostic impact was more pronounced in the intermediate cytogenetic risk group (EFS; HR, 1.64; P < .001; and RFS; HR, 1.55; P = .02), and was also apparent in cytogenetically normal AML (EFS; HR, 1.67; P = .008). Besides inv(3)/t(3;3), EVI1(+) was significantly associated with chromosome abnormalities monosomy 7 and t(11q23), conferring prognostic impact within these two cytogenetic subsets. EVI1(+) was virtually absent in favorable-risk AML and AML with NPM1 mutations. Patients with EVI1(+) AML (n = 28) who received allogeneic stem cell transplantation in first CR had significantly better 5-year RFS (33% +/- 10% v 0%). CONCLUSION EVI1 expression in AML is unequally distributed in cytogenetic subtypes. It predicts poor outcome, particularly among intermediate cytogenetic risk AML. Patients with EVI1(+) AML may benefit from allogeneic transplantation in first CR. Pretreatment EVI1 screening should be included in risk stratification.