Residual Hematopoietic Stem Cells Research Articles

Contribution of mutant HSC clones to immature and mature cells in MDS and CMML, and variations with AZA therapy

AbstractMyelodysplastic neoplasms (MDSs) and chronic myelomonocytic leukemia (CMML) are clonal disorders driven by progressively acquired somatic mutations in hematopoietic stem cells (HSCs). Hypomethylating agents (HMAs) can modify the clinical course of MDS and CMML. Clinical improvement does not require eradication of mutated cells and may be related to improved differentiation capacity of mutated HSCs. However, in patients with established disease it is unclear whether (1) HSCs with multiple mutations progress through differentiation with comparable frequency to their less mutated counterparts or (2) improvements in peripheral blood counts following HMA therapy are driven by residual wild-type HSCs or by clones with particular combinations of mutations. To address these questions, the somatic mutations of individual stem cells, progenitors (common myeloid progenitors, granulocyte monocyte progenitors, and megakaryocyte erythroid progenitors), and matched circulating hematopoietic cells (monocytes, neutrophils, and naïve B cells) in MDS and CMML were characterized via high-throughput single-cell genotyping, followed by bulk analysis in immature and mature cells before and after AZA treatment. The mutational burden was similar throughout differentiation, with even the most mutated stem and progenitor clones maintaining their capacity to differentiate to mature cell types in vivo. Increased contributions from productive mutant progenitors appear to underlie improved hematopoiesis in MDS following HMA therapy.

Clinical Response to Azacytidine (AZA) Is Associated with Increased Contribution from Mutated Blood Progenitors: Insights from Single Cell Genotyping of Matched Stem/Progenitor and Mature Blood Cells from MDS/CMML Patients Pre- and Post-AZA Treatment

Introduction: Myelodysplastic Syndrome (MDS) and Chronic Myelomonocytic Leukemia (CMML) are clonal disorders driven by progressively acquired somatic mutations in hematopoietic stem cells (HSC) and characterized by the accumulation of blasts in the bone marrow and accompanying cytopenias. Hypomethylating agents (HMA) such as azacytidine (AZA) can modify the clinical course of MDS and CMML (Platzbecker et al Blood 2019). Although not curative, they are used as first-line therapies for high-risk patients who are ineligible or unable to access an allogeneic bone marrow transplant. Clinical improvement in response to HMAs is not accompanied by clearance or major shifts in mutant clones in the bone marrow (Unnikrishnan et al Cell Reports 2017, Merlevede et al Nature Communications 2016). However, it is unclear whether in patients with established MDS; (a) hematopoietic stem and progenitor cells with multiple mutations progress through to mature cells with comparable robustness to their counterparts with fewer or no mutations, or (b) the improvements in peripheral blood counts following HMA therapy is driven by residual wild-type hematopoietic stem and progenitor cells or by clones with particular combinations of mutations. Methods: We index sorted hematopoietic stem (HSC, MPP) and progenitor cells (CMP, MEP and GMP) and mature blood cells (neutrophils (Neut), monocytes (Mono), and naïve B-cells (nBC): short lived cell types representative of current stem/progenitor output) from 3 MDS/CMML patients (1 treatment naïve, 2 treated >10 years with AZA) and performed targeted amplicon sequencing on thousands of single cells (n = 4248). In a second cohort (9 MDS patients; 6 responders, 3 non-responders) we sorted Mono, natural killer (NK) cells, and CD33+ progenitors and measured variant allele fraction (VAF) before and after 6 cycles of AZA. Results: Single cell data showed the proportion of residual wild-type HSCs, and their contribution to mature myeloid cells, was minor (0 - 5.3% of HSCs, 0.3 - 4.5% of mature myeloid cells). Driver mutations were proportionately represented across multiple hematopoietic cell types, and even the most mutated stem and progenitor clones maintained their capacity to differentiate to mature myeloid and, in some cases, lymphoid cell types in vivo, irrespective of AZA treatment (Fig A). In a second cohort composed of paired pre- and post-AZA samples, clonal composition differed slightly between cell types; e.g. NK cells often harbored lower VAFs compared to monocytes and progenitors. We also observed treatment-associated reduction in a minority of mutated clones in this cohort. However, in all patients, clonal composition was remarkably similar before and after treatment, with highly mutated progenitors making a significant contribution to mature cells, even in patients showing reduced blast plus improved blood counts (Fig B). Conclusions: Highly mutated immature cells contribute significantly to mature blood production in MDS and CMML, before and after AZA treatment. Our data highlight the key role of AZA therapy to promote output from mutant progenitors rather than simply eradicating them. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting IDH1-Mutated Pre-Leukemic Hematopoietic Stem Cells in Myeloid Disease, Including CCUS and AML

Introduction: Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 have been reported in clonal hematopoiesis and are recurrent, early events in the development of acute myeloid leukemia (AML). A subsequent stepwise acquisition of additional genetic aberrations leads to the development of frank leukemia. At the time of AML diagnosis, rare pre-leukemic hematopoietic stem cells (pHSC) can be detected containing some, but not all, of the mutations in the corresponding leukemia, although these pHSCs are phenotypically indistinguishable from residual wildtype hematopoietic stem cells (HSC). The frequency of pHSCs at the time of diagnosis is known to correlate to overall survival in AML patients (Corces et al, Nat Genet 2016), indicating the potential relevance of therapeutically targeting these cells to prevent relapse. Methods: To model these rare pre-leukemic cells, we introduced IDH1 R132H and IDH2 R140Q mutants or their wildtype counterparts into human CD34+ hematopoietic stem and progenitor cells (HSPC) using CRISPR/Cas9 and AAV6-mediated homology directed repair (HDR). Successfully edited cells were then sorted by FACS based on GFP expression for subsequent analyses including 2HG quantification, colony formation, differentiation capacity, RNA sequencing, and metabolic assays. Additionally, primary pHSCs from diagnostic bone marrow aspirates from AML patients were also isolated by FACS and subjected to treatment, colony formation assays, and genotyping by ddPCR. Results: Human CD34+ HSPCs engineered to express either IDH1 R132H or IDH2 R140Q produced 2HG and showed a striking reduction in colony formation and differentiation capacity, recapitulating the cytopenic phenotype observed in IDH-mutated clonal cytopenia of undetermined significance (CCUS). RNA-seq of these cells revealed large transcriptomic changes shared between IDH1 R132H and IDH2 R140Q mutations, but gene set enrichment analyses revealed the specific and significant downregulation of oxidative phosphorylation-related genes in R132H-mutated cells. These R132H cells were further confirmed to have lower basal metabolic activity coupled to a higher mitochondrial dependency as determined by SCENITH (Argüello et al, Cell Metab 2021). These metabolic alterations suggested that it might be possible to selectively therapeutically target R132H mutated cells. To evaluate this, we treated HSPCs engineered to express GFP, IDH1 wildtype, or IDH1 R132H, with the known mutant-specific inhibitor ivosidenib, the OXPHOS-inhibitor IACS-010759, or DMSO as a control. Strikingly, ivosidenib restored the defect in colony formation conferred by R132H and increased the total number of colonies whereas IACS-010759 effectively inhibited R132H colony formation (Figure 1A). To further investigate this vulnerability, we sorted pHSCs from diagnostic bone marrow samples from primary IDH1 and IDH2-mutated AML samples according to the phenotype CD45dimCD3-CD19-CD20-CD34+CD38-CD99-TIM3-. These cells were subjected to treatment with DMSO, ivosidenib, enasidenib, or IACS-010759 and subsequently plated for colony formation. Because the sorted cell population contained a mixture of mutant pHSCs and residual wildtype HSCs, all colonies were picked and genotyped for IDH mutations using ddPCR, revealing that only IDH1 wildtype colonies grew out after IACS-010759 treatment whereas all other conditions resulted in a mixture of wildtype and mutant colonies (Figure 1B). Conclusions: The OXPHOS-inhibitor IACS-010759 can eradicate IDH1-mutated pHSCs whereas ivosidenib cannot. This effect is specific for IDH1 mutations and the same effect is not seen in IDH2-mutated pHSCs. These results have potential clinical implications when considering therapeutic options in AML as well as IDH1-mutated pre-leukemic conditions such as CCUS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Evaluating Stem Cells to Predict Post-Transplant Relapse in the Myelodysplastic Syndromes

Malignant hematopoietic stem cells (HSCs) both initiate the myelodysplastic syndromes (MDS) and drive treatment failure through their resistance to frontline non-transplant therapies. Consequently, allogeneic transplant is the only curative therapy, but relapse can still occur. While post-transplant relapse is hypothesized to be driven by persistent MDS HSCs, their detection has proven challenging due to their limiting frequency in biospecimens. The accuracy of sequencing low-input specimens is limited by allelic drop-out and amplification bias. We adapted a hybridization capture-based targeted sequencing panel covering 260 myeloid malignancy associated genes that incorporates unique molecular identifiers capable of retaining read depths of >12x from as few as 19 sorted HSCs. We aimed to characterize residual disease after transplant with greater resolution than previously possible by sequencing sorted CD34+CD38-CD45RA- HSCs and multipotent progenitors (MPPs), CD34+CD38-CD45RA+ lymphoid-primed multipotent progenitors (LMPPs), and CD34+CD38+ progenitors. We applied our technique to an MDS case characterized by bi-allelic TP53 mutations (Y220C and P75fs), assessing a bone marrow (BM) specimen D+168 after transplant, which was five months prior to clinical relapse. Both mutations were present at variant allelic frequencies (VAFs) of 8% and 4% in 258 sorted CD34+CD38- cells with 105x coverage (xc), but simultaneously undetectable in bulk BM (481xc) and CD34+CD38+ progenitor cells (239xc) despite greater cell number and read depth. We next evaluated a D+179 post-transplant BM from a case of MDS harboring ASXL1 (R713G), DNMT3A (R882H), and IDH2 (R140Q) mutations. All three mutations were detectable in 102 sorted HSCs/MPPs at VAFs of 20% (92xc), 13% (68xc), and 35% (89xc), respectively, while absent in bulk BM (369xc) and CD34+CD38+ progenitors (506xc). These findings preceded clinical relapse by nearly two years. We additionally investigated pre- and post-transplant BM samples of a patient with a germline DDX41 M1I mutation treated for MDS. In such cases, a second somatic DDX41 mutation is often acquired, impairing ribosomal biogenesis. This mutation is always present at a low VAF, and it is unclear how it promotes disease. Prior to transplant, we identified a somatic DDX41 R525H mutation at a VAF of 2.6% in bulk BM. However, this mutation was present at a VAF of 45% in sorted HSCs/MPPs, revealing the pervasiveness of this "minor clone” at the apex of the hematopoietic hierarchy (Fig. 1). Additionally, two previously unknown TP53 clones were discovered at VAFs of 22% (K132R) and 1.8% (C275F) in HSCs/MPPs, and these were undetectable in bulk BM. At D+102 post-transplant, we detected persistence of both M1I (VAF 43%) and R52H (VAF 27%) DDX41 mutations in 19 sorted HSCs/MPPs (12xc) 78 days prior to relapse. These data suggest that while impaired ribosomal biogenesis in HSCs with bi-allelic DDX41 mutations prevents effective hematopoiesis, it also increases self-renewal and accumulation of HSCs. Next, we sought to test if early identification of residual MDS HSCs post-transplant is predictive of disease relapse. We evaluated a cohort of patients who underwent myeloablative allogeneic transplant for MDS at UTSW and MSKCC (n=21). We evaluated BMs available from post-transplant dates ranging from D+30 to D+120, including any specimen from which we were able to sort at least 15 HSCs/MPPs and achieve an effective read depth of >10. We censored specimens with follow-up of less than one year. With a median follow-up of two years, detection of disease mutations in HSCs/MPPs was 100% specific and 88% sensitive for relapse (Fig. 2). The average time from detection of disease in HSCs to clinical relapse was 6.9 months. Detection in CD38+CD34+ progenitors was also 100% specific but only 67% sensitive for relapse. Assessment of mutations in bulk BM (mean 529xc) was only 22% sensitive and 50% specific for relapse. In conclusion, our research shows for the first time that relapse of MDS after allogeneic transplant is driven by failure to eradicate MDS HSCs, and that detection of MDS HSCs early after transplant is highly predictive for relapse. Validation of this assay in larger cohorts promises to yield a tool to identify patients who may benefit from early post-transplant interventions to forestall relapse. We also demonstrate how this assay can reveal novel insights into human disease HSC biology. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Understanding of the crosstalk between normal residual hematopoietic stem cells and the leukemic niche in acute myeloid leukemia

Acute myeloid leukemia (AML) is a heterogeneous disease, yet clinically most patients present with pancytopenia resulting from bone marrow failure, predisposing them to life-threatening infections and bleeding. The mechanisms by which AML mediates hematopoietic suppression is not well known. Indeed, much effort has so far been focused on how AML remodels the bone marrow niche to make it a more permissive environment, with less focus on how the remodeled niche affects normal hematopoietic cells. In this perspective, we present evidence of the key role of the bone marrow niche in suppressing hematopoietic stem cells (HSCs) during leukemic progression and provide perspectives on how future research on this topic may be exploited to provide treatments for one of the key complications of AML.

3089 – ELUCIDATING THE MECHANISM OF ACTION OF INTERFERON-Α THERAPY IN THE TREATMENT OF MYELOPROLIFERATIVE NEOPLASMS

The majority of patients with Bcr-Abl negative myeloproliferative neoplasms (MPN) have a JAK2-V617F mutation. JAK-inhibitors have been shown to normalize blood count and reduce splenomegaly, but does not substantially reduce the mutant allele burden. Interferon-alpha (IFNα) treatment is currently the only option for MPN patients that achieves molecular remission. However, long-term treatment with IFNα is required to achieve this and patients frequently exhibit adverse side-effects. Thus it is important to understand the mechanism of action of IFNα in achieving molecular remission. Previous studies in JAK2-mutant MPN mouse model have shown that IFNα treatment leads to the depletion of the mutant hematopoietic stem cells (HSCs). Our previous findings have shown that IFNα can drive wildtype dormant HSCs into cycling and leads to attrition of HSC function. To assess the inherent proliferative index of MPN-stem cells and the effect of IFNα treatment on mutant-HSC dormancy, a BrdU pulse-chase experiment was performed on a JAK2 mutant mouse model. The kinetics of BrdU label loss was assessed and the label-dilution was observed to be significantly faster in the JAK2-mutant HSCs compared to wildtype HSCs. This suggests that the mutant HSCs have an inherently high cycling compared to wildtype HSCs. However, BrdU-high residual label-retaining mutant-HSCs were observed even at 20 weeks post-induction of mutation. This suggests that a sub-fraction of mutant-HSCs stay quiescent and might contribute as a reservoir of mutant HSCs that propagate the disease. The mutant mice will be further treated with IFNα, post-labelling with BrdU, to assess if the JAK2-mutant HSCs can be further driven into cycle more than wild-type HSCs. The proportion of residual label-retaining HSCs will also be analyzed following JAK-inhibitor treatment.

Mesenchymal niche remodeling impairs hematopoiesis via stanniocalcin 1 in acute myeloid leukemia

Acute myeloid leukemia (AML) disrupts the generation of normal blood cells, predisposing patients to hemorrhage, anemia, and infections. Differentiation and proliferation of residual normal hematopoietic stem and progenitor cells (HSPCs) are impeded in AML-infiltrated bone marrow (BM). The underlying mechanisms and interactions of residual hematopoietic stem cells (HSCs) within the leukemic niche are poorly understood, especially in the human context. To mimic AML infiltration and dissect the cellular crosstalk in human BM, we established humanized ex vivo and in vivo niche models comprising AML cells, normal HSPCs, and mesenchymal stromal cells (MSCs). Both models replicated the suppression of phenotypically defined HSPC differentiation without affecting their viability. As occurs in AML patients, the majority of HSPCs were quiescent and showed enrichment of functional HSCs. HSPC suppression was largely dependent on secreted factors produced by transcriptionally remodeled MSCs. Secretome analysis and functional validation revealed MSC-derived stanniocalcin 1 (STC1) and its transcriptional regulator HIF-1α as limiting factors for HSPC proliferation. Abrogation of either STC1 or HIF-1α alleviated HSPC suppression by AML. This study provides a humanized model to study the crosstalk among HSPCs, leukemia, and their MSC niche, and a molecular mechanism whereby AML impairs normal hematopoiesis by remodeling the mesenchymal niche.

Leukemic Stress Targets the mTOR Pathway to Suppress Residual HSC in the BM Microenvironment

The remodeling of the bone marrow (BM) microenvironment that occurs along with the progressive spread of acute myeloid leukemia (AML) cells can be considered a constitutive aspect of leukemogenesis. To date most studies have focused on the functional and in part inflammatory adaptation of stroma, and its potential role in extrinsic chemotherapy resistance. Much less is known about the impact of leukemic stress on residual hematopoietic cells. We previously identified the trafficking of select microRNAs (miRs-) in extracellular vesicles (EVs) between AML cells and hematopoietic progenitor cells (HPC). These studies revealed the mechanism underlying the suppression of HPC function in the AML niche (Hornick, Doron et. al., Science Signaling 2016). Several groups, including ours also noted the relative resistance of residual hematopoietic stem cells (HSC) to elimination from the BM of xenografted animals. In the current study we set out to understand how leukemic stress in the AML xenograft niche shapes HSC fate and function. Using AML cell lines (Molm14, U937, HL60) we established NSG xenografts, systematically tracking peripheral blood AML chimerism to recover murine hematopoietic cells at low or negative tumor burden, and replicating key assays using purified EV for intrafemoral injections. In immunofluorescent studies we initially confirmed the uptake of GFP labeled xenograft-derived EVs across the spectrum of HPC and HSC (KSL/CD150+/CD48-), as well as the successive loss and peripheral displacement of HPCs, and gains in HSC frequency in the leukemic niche. These HSC were found to be enriched for G0 cell cycle status with an increase in phospho- p53, but showed no evidence of apoptosis or senescence. To understand the mechanism underlying their apparent quiescence, we performed in vitro proteomics studies of AML EV exposed HPSC identified downregulation of ribosomal biogenesis pathways. We then confirmed in vivo that residual HSC from AML xenografts experienced a loss of protein synthesis (OPP assay). We next reasoned that deficits in ribosome dysfunction and protein synthesis may reflect deregulation by specific miRNAs highly abundant in AML EV. Here, we had an opportunity to profile EV miRNA from the plasma of 12 unselected AML patients at diagnosis versus 12 control samples, and we confirmed a significant enrichment for specific miRNAs, including miR-1246. Raptor is a component of the mTOR pathway and an annotated target of miR-1246. We demonstrated in a series of experiments that miR-1246 translationally suppresses Raptor and downregulates protein synthesis in residual HSC from AML xenografts. The transfection of synthetic anti-miR1246 sequences on the other hand reversed the effects of AML EV in murine HSC. In aggregate we show that direct crosstalk between AML and hematopoietic cells adds to the adaptive changes that occur in the AML niche. Our experiments suggest a functional significance for EV miRNA that can be detected in AML patient plasma in the regulation of residual BM HSC. More broadly, the mechanisms by which leukemic stress alters hematopoietic function remain underexplored, but our observations suggest that leukemia derived EV contribute to changes in competitive fitness of residual HSC. Disclosures No relevant conflicts of interest to declare.

Somatic Mutations Potentiating RAS-MAPK Signaling Confer Resistant Potential Against FLT3-Inhibitors to Acute Myelogenous Leukemia

Acute myeloid leukemia (AML) is one of the most common hematologic malignancies derived from a small number of self-renewing leukemic stem cells (LSCs). AML has various genetic abnormalities such as chromosomal aberration, single nucleotide variant and insertion/deletion. In particular, internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3-ITD) is the most frequently found in ~ 25 % of AML cases, moreover, AML with FLT3-ITD is classified in unfavorable. FLT3 is fundamentally essential for survival of hematopoietic stem cells (HSCs) as well as LSCs, but once ITD is acquired, FLT3-ITD promotes the proliferation of AML by activating downstream enzymes. Various FLT3 inhibitors are now available as effective and promising drugs. On the other hand, the resistance against those inhibitors has been regarded as an emerging problem in clinics. To clarify the molecular machineries how AML cells acquire the resistance against FLT3 inhibitors, we compared the mutation status of paired AML samples consisted of diagnostic and relapsed samples. LSCs, which are quiescent and highly resistant against conventional chemotherapies, are considered to play a central role in recurrence. Since we and other group previously reported that TIM-3 clearly discriminates LSCs from residual normal HSCs, we purified and evaluated the mutations of CD34+CD38-TIM-3+ LSCs or CD34+ immature AML cells. The paired diagnostic and relapsed samples obtained from the identical 5 AML patient treated with Gilteritinib were subjected to whole exome sequencing (WES) analysis. CD3+ T cells were also purified from the diagnostic samples and used as a germ line control to detect somatic mutations acquired in AML cells. Libraries for WES were prepared using Sure Select XT HS and Human All Exon V6 Capture Kit in accordance with manufacturer's instructions. Then we performed WES using NextSeq 500 with 150bp pair-end read. Surprisingly, in all 5 cases examined, AML clones lost FLT3-ITD, whereas clones harboring mutations potentiating RAS-MAPK signaling became dominant at relapse: one case acquired the activated KRAS (Q61H) mutation and the remaining four cases newly acquired the previously-unidentified mutations in RAS-MAPK inhibitory genes. Such mutations in RAS-MAPK-inhibitory genes include DIRAS3 (W173X), RASA1 (E70G), RAP1GAP2 (K18X), RIN2 (S355fs), RASA4 (C655R), RASA4B (E141G), MAP2K7 (T66P) and PTPN7 (T14M). Of note, these genes belong to RAS-MAPK-inhibitory pathways and the encoded proteins are reported to suppress the activity of RAS-MAPK pathways. These RAS-MAPK-inhibitory mutations were exclusively detected in the relapsed AML clones, and contained stop-gain and frameshift mutations. We, therefore, hypothesized that RAS/MAPK activation driven by somatic mutations contributes to the acquisition of resistance against FLT3 inhibitors in LSCs. Consistent with such hypothesis, phosflow analysis revealed that basal ERK phosphorylation was retained or rather increased in the relapse CD34+ AML cells lacking FLT3-ITD, indicating that RAS-MAPK activation was maintained independent of FLT3-ITD. Furthermore, ASP2215 and AC220 inhibited ERK phosphorylation in the diagnostic CD34+ AML cells, whereas both inhibitors failed in the inhibition of ERK activation in the relapsed AML clones in all 3 cases analyzed. Gilteritinib also failed in inhibiting ERK in other relapsed AML cases, however, the combination of ASP2215 and MEK inhibitor efficiently suppressed ERK phosphorylation. All of relapse AML cases exhibited the emergence of AML clones harboring mutations associated with RAS-MAPK pathway genes, and therefore, the clinical sequence for detecting such mutations might be useful to assess the risk of FLT3-inhibitor treatment failure in clinics. Furthermore, our study showed the efficacy of MEK inhibitors to inhibit RAS-MAPK signaling activity even in the relapsed AML cells. Thus, the combination therapy consisted of FLT3-inhibitors and MEK inhibitors might be one of the promising therapeutic strategies to overcome FLT3-inhibitor resistance in AML. In summary, our study revealed that mutations potentiating RAS-MAPK pathway should play a central role in conferring the resistance against FLT3-inhibitors to AML. Disclosures Akashi: Sumitomo Dainippon, Kyowa Kirin: Consultancy; Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding.

Phenotypic characterization of malignant progenitor cells in patients with idiopathic myelofibrosis

Objective/BackgroundIdiopathic myelofibrosis (IM) is a clonal hematological malignancy originating from pluripotent hematopoietic stem cells (HSC). HSC are very rare potent cells that reside in the bone marrow (BM) and at a lower level in peripheral blood (PB). Previous studies showed that IM PB CD34+ cells contain not only BM repopulating cells belonging to the malignant clone but also residual normal HSC. MethodsIn the current study, we separated the subpopulations of IM PB CD34+ cells using IL-3Rα/CD123 labeling and further characterized them by genetic and functional analyses. ResultsWe differentiated IM PB CD34+ cells into three subpopulations (IL-3Rαhigh, IL-3Rαlow, and IL-3Rαnegative). IL-3Rαhigh CD34+ cell subgroup represents a small population in IM PB CD34+ cells which was not seen in normal G-CSF mobilized CD34+ cells. IM IL-3Rαhigh CD34+ cells contained significant higher percentage of cells bearing marker chromosome detected by fluorescence in situ hybridization (FISH) analysis. In the absence of growth factors, IM IL-3Rαhigh CD34+ cells exhibited abnormal colony forming ability and carried greater percentage of JAK2V617F mutant allele compared with IL-3Rαlow and IL-3Rαnegative CD34+ cells. ConclusionThese data indicate that IL-3Rαhigh CD34+ cells from IM enriched for the malignant progenitor cells and IL-3Rα/CD123 may be a potential biomarker and therapeutic target for IM. Our findings will be further validated in future studies with a larger sample size and serial transplant in murine models.

Megakaryocyte-derived excessive transforming growth factor β1 inhibits proliferation of normal hematopoietic stem cells in acute myeloid leukemia

Impaired production of healthy hematopoietic cells from residual hematopoietic stem cells (HSCs) leads to high mortality in acute myeloid leukemia (AML). Previous studies have identified p21 and Egr3 as intrinsic factors responsible for the growth arrest and differentiation blockade of normal HSCs in leukemia; however, the related extrinsic factors remain unknown. In this study, we found that transforming growth factor β (TGFβ) signaling was upregulated in HSCs from bone marrow of mice with MLL-AF9-induced acute myeloid leukemia (AML) because of excessive production of TGFβ1, especially from megakaryocytes, and overactivation of latent TGFβ1 protein. We also found that SMAD3, a signal transducer of TGFβ1, directly bound to Egr3 and upregulated its expression to arrest proliferation of HSCs. Our study provides evidence for targeting TGFβ1 in AML to rectify normal hematopoiesis defects in clinical practice.

Forme pseudo-tumorale de la tuberculose osseuse chez l’enfant : à propos d’une localisation humérale rare

Impaired production of healthy hematopoietic cells from residual hematopoietic stem cells (HSCs) leads to high mortality in acute myeloid leukemia (AML). Previous studies have identified p21 and Egr3 as intrinsic factors responsible for the growth arrest and differentiation blockade of normal HSCs in leukemia; however, the related extrinsic factors remain unknown. In this study, we found that transforming growth factor β (TGFβ) signaling was upregulated in HSCs from bone marrow of mice with MLL-AF9-induced acute myeloid leukemia (AML) because of excessive production of TGFβ1, especially from megakaryocytes, and overactivation of latent TGFβ1 protein. We also found that SMAD3, a signal transducer of TGFβ1, directly bound to Egr3 and upregulated its expression to arrest proliferation of HSCs. Our study provides evidence for targeting TGFβ1 in AML to rectify normal hematopoiesis defects in clinical practice.

The bulk of the hematopoietic stem cell population is dispensable for murine steady-state and stress hematopoiesis

AbstractLong-term repopulating (LT) hematopoietic stem cells (HSCs) are the most undifferentiated cells at the top of the hematopoietic hierarchy. The regulation of HSC pool size and its contribution to hematopoiesis are incompletely understood. We depleted hematopoietic stem and progenitor cells (HSPCs) in adult mice in situ and found that LT-HSCs recovered from initially very low levels (<1%) to below 10% of normal numbers but not more, whereas progenitor cells substantially recovered shortly after depletion. In spite of the persistent and massive reduction of LT-HSCs, steady-state hematopoiesis was unaffected and residual HSCs remained quiescent. Hematopoietic stress, although reported to recruit quiescent HSCs into cycle, was well tolerated by HSPC-depleted mice and did not induce expansion of the small LT-HSC compartment. Only upon 5-fluorouracil treatment was HSPC-depleted bone marrow compromised in reconstituting hematopoiesis, demonstrating that HSCs and early progenitors are crucial to compensate myeloablation. Hence, a contracted HSC compartment cannot recover in situ to its original size, and normal steady-state blood cell generation is sustained with <10% of normal LT-HSC numbers without increased contribution of the few residual cells.

The bulk of the hematopoietic stem cell population is dispensable for murine steady-state and stress hematopoiesis

Long-term repopulating (LT) hematopoietic stem cells (HSCs) are the most undifferentiated cells at the top of the hematopoietic hierarchy. The regulation of HSC pool size and its contribution to hematopoiesis are incompletely understood. We depleted hematopoietic stem and progenitor cells (HSPCs) in adult mice in situ and found that LT-HSCs recovered from initially very low levels (<1%) to below 10% of normal numbers but not more, whereas progenitor cells substantially recovered shortly after depletion. In spite of the persistent and massive reduction of LT-HSCs, steady-state hematopoiesis was unaffected and residual HSCs remained quiescent. Hematopoietic stress, although reported to recruit quiescent HSCs into cycle, was well tolerated by HSPC-depleted mice and did not induce expansion of the small LT-HSC compartment. Only upon 5-fluorouracil treatment was HSPC-depleted bone marrow compromised in reconstituting hematopoiesis, demonstrating that HSCs and early progenitors are crucial to compensate myeloablation. Hence, a contracted HSC compartment cannot recover in situ to its original size, and normal steady-state blood cell generation is sustained with <10% of normal LT-HSC numbers without increased contribution of the few residual cells.

A giant adrenal myelolipoma in a beta-thalassemia major patient: Does ineffective erythropoiesis play a role?

A 40-year-old thalassemia major (genotype β0 cod39/β+ IVSI-110) patient, regularly transfused with three units of packed red cells every three weeks with an average pre-transfusion hemoglobin level of 100 g/L, presented in 2002 with a right abdominal mass measuring 7.5 cm of maximum width found incidentally during the annual ultrasound (US) examination of the abdomen. A subsequent MRI showed radiological features suggestive for extramedullary hematopoiesis (EMH) thus the mass was periodically monitored. In 2009, because of the progressive increase in mass volume, hydroxyurea therapy was started at increasing dosage up to 15 mg/kg per day. In 2014 and 2015, a US evaluation showed a rapidly increasing volume of the abdominal mass. A computed tomography (CT) scan of the abdomen and pelvis with intravenous iodine contrast (Image 1) showed a huge well circumscribed retroperitoneal poly-lobed mass in the right abdomen measuring 19 × 15 × 25 cm3, originating from the right adrenal gland and displacing the liver medially and the right kidney caudally and anteriorly; moreover, it compressed the inferior vena cava and displaced the mesenteric vessels. The mass was poorly vascularized with low contrast enhancement and marked heterogeneity, primarily due to the presence of fat tissue. At this time the radiological features were more suggestive of either an angiomyolipoma or a myelolipoma rather than EMH. Laparotomy was performed and a 3.5 kg encapsulated tumor (Image 1) attached to the right adrenal gland was removed. Gross examination showed a soft yellow to red cut surface with focal areas of hemorrhage. Extensive sampling was performed given the size of the mass and microscopic examination showed typical features of myelolipomas, with varying proportions of mature adipose tissue admixed with trilineage hematopoiesis (Image 1). A thin rim of residual adrenal tissue was present at the edge of the mass. Myelolipoma is a rare tumor that accounts for approximately the 4% of adrenal tumors. Some cases are associated with chronic hemolytic anemias or ineffective erythropoiesis, including both transfusion dependent and transfusion independent thalassemia 1-4, sickle cell anemia 5, 6, and hereditary spherocytosis 7, 8. In a few cases the diagnosis of myelolipoma allowed the retrospective diagnosis of the underlying congenital anemia 5, 8. These associations suggest that the tumor is under the control of hematopoietic stimuli. It has been demonstrated that the first hematopoietic activity, known as primitive hematopoiesis, appears in the blood islands of the yolk sac. However, the first hematopoietic stem cells (HSC), defined by their capacity for long-term and multi-lineage engraftment in adult recipients, appear in the mammal embryo in the (AGM) region, and then home to the fetal liver 9, 10. We demonstrated by flow cytometry that the mass of our patient contained erythroid precursors (Image 1). Hence, residual HSC in organs derived from the AGM region in patients with ineffective erythropoiesis might be the source of the tumor mass. In our cohort of 190 patients affected by transfusion dependent thalassemia, we observed four cases of myelolipoma, three originating from the adrenal glands and one from the pelvic region. Further investigations involving a greater number of patients are needed to establish the prevalence of a tumor that might be underestimated in patients affected by diseases with chronic anemia and ineffective erythropoiesis. A: Computed tomography scan with iodine contrast showing a huge, well circumscribed, poorly vascularized retroperitoneal mass in the right abdomen measuring 19 cm × 15 cm × 25 cm3, originating from the right adrenal gland, displacing the liver and the right kidney and compressing the inferior vena cava and mesenteric vessels. B: A 3.5 kg encapsulated tumor was removed after laparotomy. The mass was soft, yellow to red in color, with focal areas of hemorrhage. C, D: FACS analysis of cell surface markers of cells scraped from tumor sections were gated based on the forward scatter (FSC) parameter and the expression of CD45, CD71, and Glycophorin A (Gly A) was evaluated. Most cells did not express CD45 (C); the CD45 negative population expressed CD71 but no Glycophorin, as expected from erythroblasts (D). E: (20× magnification) Histological examination revealed a tumor composed of varying proportions of mature adipose tissue admixed with trilineage hematopoiesis, with numerous megakaryocytes. F–H: Immunohistochemical staining for myeloperoxidase (MPO; F, 20×), Glycophorin A (F, 20×) highlighted myeloid and erythroid cells, respectively; their relative proportions varied across different areas of the tumor. Staining for CD34 (H, 20×) highlighted endothelial cells but did not show a significant increase in blasts.

Eltrombopag Added to Standard Immunosuppression for Aplastic Anemia Accelerates Count Recovery and Increases Response Rates

Immunosuppressive treatment (IST) with horse antithymocyte globulin (hATG) and cyclosporine (CsA) for severe aplastic anemia (SAA) has a 60-65% rate of overall hematologic response (OR) and approximately 10% complete response (CR). These rates have remained stable over the last three decades despite efforts to improve upon this regimen (Scheinberg Blood 2012). Eltrombopag (EPAG), an oral thrombopoietin receptor agonist has activity in refractory SAA producing hematologic responses, the majority multilineage, in about 40% of patients when administered as a single agent (Olnes NEJM 2012; Desmond Blood 2014). We conducted an investigator-initiated, phase II, single-center trial to test the efficacy of adding EPAG to h-ATG/CsA (clinicaltrials.gov, NCT01623167), using a triple cohort design to optimize the regimen.Eighty-eight patients with treatment-naive SAA have been enrolled from July 2012 to October 2015. All received standard h-ATG and CsA. EPAG was administered at 150 mg daily to three consecutive cohorts: in the first (n=30) and second (n=31) cohorts, EPAG was introduced after hATG, from day 14 until 6 months (cohort 1) or 3 months (cohort 2), due to initial concern of hepatotoxicity. In the third cohort, EPAG was concurrent with hATG on day 1 and continued for 6 months (n=27). As analysis of our previous IST trials suggested that CR correlates with better survival and less evolution to MDS/AML, the primary endpoint in the current protocol was CR at 6 months.Baseline enrollment characteristics were similar across cohorts: median age was 31 years (range 3 – 82; 20% were <18 years old), 26% had very severe disease (ANC<200/uL), and 34% had PNH clones >1%. EPAG was well tolerated when combined with CsA and hATG; 2 patients discontinued EPAG for severe cutaneous reactions, and 6 patients withdrew before 6 months due to refractoriness (n=4) or evolution to MDS (n=2). OR and CR were higher at 3 months and 6 months for all cohorts compared to historic rates (Table): OR at 6 months was 80%, 87% and 92%, respectively for the three cohorts; CR at 6 months was 33%, 26%, and 54%, respectively. For all patients evaluable to date, OR at 3 and 6 months of 80% and 85% and CR rates of 28 and 34% were higher when compared to our historical rates (p<0.001 for both 3 and 6 months).Improvements in blood counts among partial responders were robust by 3 months in all cohorts: median ANC 1790/ul; hemoglobin 10 gm/dL; and platelets 60,500/ul. For patients with very severe neutropenia (ANC<200/ul) the median time to ANC >500/uL was 47 days for all cohorts (n=23), and 35 days for cohort 3 (n=7). Among responders, median time to transfusion-independence for platelets was 32 days and for red cells 42 days. Serial BM biopsies showed improved cellularity in 80% without increased fibrosis. Median increase from baseline in BM CD34+ cells, as measured by flow cytometry, was 17-fold and 4-fold at 3 months for cohorts 1 and 2 respectively (p<0.001). Pharmacokinetics at 3 months showed a trend toward higher drug bioavailability with older age and female sex. Previously established baseline predictors for response such as telomere length, reticulocyte count, lymphocyte count, paroxysmal nocturnal hemoglobinuria, were not predictive of outcome. Twelve patients underwent hematopoietic stem cell transplantation (HSCT) due to relapse (n=3), refractoriness (n=6) or evolution to MDS (n=3). Death occurred in 3 subjects.At median follow up 15 months (range 1 – 42 m), cytogenetic abnormalities have occurred in 7 patients: 4 monosomy or partial deletion of chromosome 7, 1 complex (t(3;3)(q21;q26), -7), 1 deletion 13q that later normalized, and 1 subject with trisomy 6 and trisomy 15 in 2 metaphases. We sequenced 54 candidate genes, recurrently mutated in MDS/AML: somatic mutations were not identified in 4 of 7 subjects prior to treatment or at the time of cytogenetic evolution. Clonal evolution to MDS occurred at a similar frequency compared to our historic experience with standard IST.Addition of EPAG to IST markedly increases overall and complete hematologic response rates in treatment-naive SAA. These results suggest that immediate institution of EPAG with IST may salvage and expand residual hematopoietic stem cells in aplastic anemia, accelerating the rate and quality of hematopoietic recovery. Rapid blood count improvement and reduced transfusion burden support early use of EPAG for patients with newly diagnosed severe aplastic anemia. [Display omitted] Disclosures:Townsley:Novartis: Research Funding ; GSK: Research Funding. Off Label Use: Eltrombopag (Promacta) is not FDA approved for treatment naive aplastic anemia. Dumitriu:Novartis: Research Funding ; GSK: Research Funding. Winkler:GSK: Research Funding ; Novartis: Research Funding. Larochelle:Novartis: Research Funding ; GSK: Research Funding. Dunbar:Novartis: Research Funding ; GSK: Research Funding. Young:Novartis: Research Funding ; GSK: Research Funding.

Abstract 3890: MicroRNA-551b is highly expressed in hematopoietic stem cells and expression in acute myeloid leukemia is associated with relapse and poor survival

Abstract Despite high remission rates after chemotherapy, only 30-40% of acute myeloid leukemia (AML) patients survive five years after diagnosis. The main cause for this treatment failure is insufficient eradication of a subpopulation of chemotherapy resistant leukemic cells with a stem cell-like character. These so-called leukemic stem cells (LSC) are thought to be responsible for relapse. We hypothesized that success of novel anti-AML therapies relies on functional manipulation of genes including microRNAs, resulting in elimination of leukemic (stem) cells while sparing residual co-existing normal hematopoietic stem cells (HSC). We aimed at identification of microRNAs differentially expressed between HSC, LSC and the AML bulk obtained from the same AML bone marrow, taking into account the effects of the leukemic microenvironment. To that end, we described immunophenotypic markers that can distinguish LSC from HSC. Moreover, we identified that HSC have higher aldehyde dehydrogenase activity than LSC (Schuurhuis et al. Plos One 2013). Comparing the microRNA expression profile of LSC with that of HSC showed that microRNA-551b (miR-551b) is highly expressed in residual HSC in the AML bone marrow. To determine whether miR-551b is a HSC specific microRNA we purified stem and progenitor cell subsets from normal bone marrow and showed that miR-551b is the highest expressed in the two most primitive CD34+CD38- populations i.e. CD90+CD45RA- HSC and CD90-CD45RA- multipotent progenitors. To investigate whether the expression of miR-551b is of clinical importance in AML we determined its expression in AML bone marrow samples (n=154) and showed that high miR-551b is associated with lower complete remission (CR) rates after the first cycle of induction chemotherapy, shorter relapse free survival and a worse overall survival. In line with miR-551b being a stem cell miRNA, high expression in AML was associated with an undifferentiated morphology (FAB M0). To shed more light on the functional role of miR-551b in AML we correlated the expression of miR-551b with overall gene expression in a large panel of AML patients. Many of the genes that highly correlated with miR-551b like; MLLT3, INPP4B, HTR1F, HOPX, PROM1 and others, are also present in published HSC signatures. In conclusion, miR-551b is specifically expressed in normal stem and multipotent progenitor cells and high expression in AML is associated with poor prognosis. Currently, our research focuses on the function of miR-551b in AML. Citation Format: David C. de leeuw, Fedor Denkers, Peter Valk, Iris de Rink, Ron Kerkhoven, Gerrit Jan Schuurhuis, Gert J. Ossenkoppele, Linda Smit. MicroRNA-551b is highly expressed in hematopoietic stem cells and expression in acute myeloid leukemia is associated with relapse and poor survival. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3890. doi:10.1158/1538-7445.AM2014-3890

Total body irradiation causes long-term mouse BM injury via induction of HSC premature senescence in an Ink4a- and Arf-independent manner

AbstractExposure to total body irradiation (TBI) induces not only acute hematopoietic radiation syndrome but also long-term or residual bone marrow (BM) injury. This residual BM injury is mainly attributed to permanent damage to hematopoietic stem cells (HSCs), including impaired self-renewal, decreased long-term repopulating capacity, and myeloid skewing. These HSC defects were associated with significant increases in production of reactive oxygen species (ROS), expression of p16Ink4a (p16) and Arf mRNA, and senescence-associated β-galacotosidase (SA-β-gal) activity, but not with telomere shortening or increased apoptosis, suggesting that TBI induces residual BM injury via induction of HSC premature senescence. This suggestion is supported by the finding that SA-β-gal+ HSC-enriched LSK cells showed more pronounced defects in clonogenic activity in vitro and long-term engraftment after transplantation than SA-β-gal– LSK cells isolated from irradiated mice. However, genetic deletion of p16 and/or Arf had no effect on TBI-induced residual BM suppression and HSC senescence, because HSCs from irradiated p16 and/or Arf knockout (KO) mice exhibited changes similar to those seen in HSCs from wild-type mice after exposure to TBI. These findings provide important new insights into the mechanism by which TBI causes long-term BM suppression (eg, via induction of premature senescence of HSCs in a p16-Arf–independent manner).

Expression Of Aldehyde Dehydrogenase Reflected Diverse Stem Cell Properties In Acute Myeloid Leukemia

Separation of leukemic stem cells (LSC) and residual hematopoietic stem cells (HSC) from the same individual patient with acute myeloid leukemia (AML) is essential for a proper understanding of the leukemic driving mechanisms. We have studied the role of aldehyde dehydrogenase (ALDH) for this purpose and have defined the functional properties of ALDHbright cells in specific subgroups of AML.We have examined the ALDH activity by flow cytometry in bone marrow samples (BM) from 14 healthy donors and 73 patients with de novo AML. The median frequency of cells with high ALDH activity (ALDHbright cells) in the healthy subjects was 1.92% with a range from 0.58 to 3.16%. For patients with AML, the median number of ALDHbright cells was 0.25% with a broad range from 0.004 to 33.57%. Whereas the majority of patients with AML (n = 56) had low frequencies of ALDHbright cells (median 0.11%; range 0.004 – 1.77%; defined as ALDH-low AML), 17 patients had relatively numerous ALDHbright cells (median 9.01; range 3.54 – 33.57%; defined as ALDH-numerous AML). In both groups, ALDHbright cell populations were highly enriched for CD34+CD38- cells. The ALDHbright cells derived from ALDH-low AML did not contain chromosomal and molecular aberrations characteristic of the original leukemia, and were able to induce multi-lineage hematopoiesis in NSG mouse models. Thus, genetically and functionally normal HSC could be successfully isolated in the ALDHbright subset, whereas LSC were enriched in ALDHdimCD34+CD38- subset for patients with ALDH-low AML. For 17 patients with ALDH-numerous AML, the ALDHbright subset was consistently contaminated with LSC. In clinical follow-ups, patients with ALDH-numerous AML showed resistance to induction chemotherapy and were characterized by a very poor long-term outcome that was comparable to patients with high-risk cytogenetic or molecular genetic markers. In four patients with ALDH-numerous AML we demonstrated that the ALDHbrightCD34+CD38- subset contained chemotherapy-resistant clones with repopulating ability. Furthermore, such ALDHbright cells were characterized by a lower cell-cycle activity and an increased resistance to cytarabine in comparison with ALDHdim blasts in in vitro assays.Our data have provided evidence that LSC and residual HSC can be separated using ALDH in patients with low frequencies of ALDHbright cells. In patients with ALDH-numerous AML, the ALDHbright subset is associated with leukemic features both in vitro and in animal models. Thus our data demonstrated the feasibility of appropriate comparisons of LSC versus HSC from the same patient with specific subtypes of AML and the impact of LSC properties on clinical outcome. Disclosures:Buss:Novartis: Travel support Other; Micromet/Amgen: Reimbursements for participation in a clinical study , Reimbursements for participation in a clinical study Other. Ho:Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Microrna-551b Is Preferentially Expressed In Hematopoietic Stem and Multipotent Progenitor Cells and High Expression In Acute Myeloid Leukemia Is An Independent Prognostic Factor For Relapse and Poor Survival

Over the last past years microRNAs (miRNAs) have emerged as important regulators of normal hematopoiesis. Growing evidence suggests that miRNAs that are expressed and function in hematopoietic stem cells (HSC) are especially important in the pathogenesis of acute myeloid leukemia (AML). HSC characteristics like self renewal, quiescence and the ability to overcome senescence have shown to be under control of miRNAs and contribute to the aggressiveness of AML. Recently we identified microRNA-551b (miR-551b) as a miRNA that is highly expressed in residual normal HSC in AML bone marrow. Thus far, no functional or clinical relevance has been described for miR-551b. To explore its clinical impact we here investigate the expression of miR-551b in highly enriched stem and progenitor cell populations in normal bone marrow and in a cohort of AML patients.Normal bone marrow showed highest expression of miR-551b in the two most primitive CD34+CD38- populations i.e. CD90+CD45RA- HSC and CD90-CD45RA- multipotent progenitor cells. In the more differentiated progenitor and mature cell populations, miR-551b expression was strongly reduced suggesting its involvement in early hematopoiesis (Figure A). To investigate whether miR-551b is expressed and of prognostic importance in AML we performed qRT-PCR expression analysis in well defined de novo AML bone marrow samples (n=154). High miR-551b expression was associated with lower complete remission (CR) rates after first-line standard-dose remission-induction therapy (p=.015) and shorter relapse free survival (RFS; p=.003) and overall survival (OS; p<.005) (Figure B/C). Also in cytogenetically normal AML high expression was associated with poor outcome (OS p=.02 and RFS p=.009). Combining miR-551b expression at diagnosis with minimal residual disease (MRD) detection after the second cycle of chemotherapy improved the prognostic value of MRD detection. Patients who were positive for MRD and/or highly expressed miR-551b showed shorter OS (p=.01) and RFS (p=.001) compared to patients who were both MRD negative and had low miR-551b expression. Moreover, in the group of MRD negative patients a higher chance of relapse was found if patients expressed miR-551b highly compared to those with low expression (66.7 vs. 27.6%). In multivariate analysis, high miR-551b expression remained an independent predictor for overall survival (p=.049) and relapse free survival (p=.001). In line with miR-551b being an early hematopoietic miRNA, high expression was correlated with more undifferentiated morphology. To shed more light on the biological role of miR-551b we correlated its expression to mRNA sequencing data in a large publically available dataset [Ley et al., NEJM 2013]. Many of the genes that highly correlated with miR-551b like: MLLT3, INPP4B, HTR1F, HOPX, PROM1 and others, are also present in published HSC gene signatures. Gene ontology analysis of the correlated genes showed significant enrichment for genes involved in hematopoiesis and cell adhesion.Our results show that miR-551b is a HSC specific miRNA and that high expression in AML is associated with a poor prognosis possibly because it reflects a stem cell-like state. Currently our research focuses on the functional role of miR-551b in normal and malignant hematopoiesis.▪ Disclosures:No relevant conflicts of interest to declare.