bims-tremyl Biomed News
on Therapy resistance biology in myeloid leukemia
Issue of 2022‒03‒20
28 papers selected by
Paolo Gallipoli
Barts Cancer Institute, Queen Mary University of London

  1. Cytometry B Clin Cytom. 2022 Mar 15.
      BACKGROUND: Myelodysplastic syndromes (MDS) at risk of transformation to acute myeloid leukemia (AML) are difficult to identify. The bone marrows of MDS patients harbor specific hematopoietic stem and progenitor cell (HSPC) abnormalities that may be associated with sub-types and risk-groups. Leukemia-associated characteristics of such cells may identify MDS patients at risk of progression to AML and provide insight in the pathobiology of MDS.METHODS: Bone marrow samples from healthy donors (n = 10), low risk (n = 12) and high risk (n = 13) MDS patients were collected, in addition, AML samples for 5 out of 6 MDS patients that progressed. Mass cytometry was applied to assess expression of stem cell subset and leukemia-associated immunophenotype markers.
    RESULTS: We analyzed the data using FlowSOM to cluster cells with similar expression of 10 commonly used stem cell markers. Metaclusters (n = 20) of these clusters represented populations of cells with a related phenotype, largely resembling known stem cell subsets. Within specific subsets, intra-cellular expression levels of pCREB, IkBα, or pS6 differed significantly between healthy bone marrow (HBM) and MDS or consecutive secondary AML samples. CD34, CD44, and CD49f expression was significantly increased in high risk MDS and AML-associated metaclusters. We identified MDS/sAML cells with aberrant phenotypes when compared to HBM. Such cells were observed in clusters of both primary MDS and secondary AML samples.
    CONCLUSIONS: High-dimensional mass cytometry and computational data analyses enabled characterization of HSPC subsets in MDS and identification of leukemia stem cell populations based on their immunophenotype. Stem cells in MDS that display leukemia-associated features may predict the risk of developing AML.
    Keywords:  acute myeloid leukemia; leukemic transformation; mass cytometry; myelodysplastic syndromes; stem cells
  2. Blood. 2022 Mar 14. pii: blood.2021013925. [Epub ahead of print]
      Myeloproliferative neoplasms (MPN) transform to myelofibrosis (MF) and highly lethal acute myeloid leukemia (AML), although actionable mechanisms driving progression remain elusive. Here, we elucidate the role of the High Mobility Group A1 (HMGA1) chromatin regulator as a novel driver of MPN progression. HMGA1 is up-regulated in MPN with highest levels after transformation to MF or AML. To define HMGA1 function, we disrupted gene expression via CRISPR/Cas9, short hairpin RNA, or genetic deletion in MPN models. HMGA1 depletion in JAK2V617F AML cell lines disrupts proliferation, clonogenicity, and leukemic engraftment. Surprisingly, loss of just a single Hmga1 allele prevents progression to MF in JAK2V617F mice, decreasing erythrocytosis, thrombocytosis, megakaryocyte hyperplasia, and expansion of stem and progenitors, while preventing splenomegaly and fibrosis within the spleen and bone marrow. RNA- and chromatin immunoprecipitation-sequencing revealed HMGA1 transcriptional networks and chromatin occupancy at genes that govern proliferation (E2F, G2M, mitotic spindle) and cell fate, including the GATA2 master regulatory gene. Silencing GATA2 recapitulates most phenotypes observed with HMGA1 depletion whereas GATA2 re-expression partially rescues leukemogenesis. HMGA1 transactivates GATA2 through sequences near the developmental enhancer (+9.5), increasing chromatin accessibility and recruiting active histone marks. Further, HMGA1 transcriptional networks, including proliferation pathways and GATA2, are activated in human MF and MPN leukemic transformation. Importantly, HMGA1 depletion enhances responses to the JAK2 inhibitor, ruxolitinib, preventing MF and prolonging survival in murine models of JAK2V617F AML. These findings illuminate HMGA1 as a key epigenetic switch involved in MPN transformation and promising therapeutic target to treat or prevent disease progression.
  3. Haematologica. 2022 Mar 17.
      Chemotherapy is the primary treatment option for acute myeloid leukemia (AML), but leukemic stem cells (LSCs) can survive chemotherapy for disease recurrence and refractory. Here, we found that AML cells obtained from relapsed patients had increased autophagy levels than de novo AML cells. Furthermore, Doxorubicin (DOX) treatment stimulated autophagy in LSCs by repressing the mTOR pathway, and pharmaceutical inhibition of autophagy rendered chemoresistant LSCs sensitive to DOX treatment in MLL-AF9 induced murine AML. Moreover, we developed a self-assembled leucine polymer, which activated mTOR to inhibit autophagy in AML cells by releasing leucine. The leucine polymer loaded DOX (Leu-DOX) induced much less autophagy but more robust apoptosis in AML cells than the DOX treatment. Notably, the leucine polymer and Leu-DOX were specifically uptaken by AML cells and LSCs but not by normal hematopoietic cells and hematopoietic stem/progenitor cells in the bone marrow. Consequently, Leu-DOX efficiently reduced LSCs and prolonged the survival of AML mice, with limited myeloablation and tissue damage side effects than DOX treatment. Overall, we proposed that the newly developed Leu-DOX is an effective autophagy inhibitor and an ideal drug to efficiently eliminate LSCs, thus serving as a revolutionary strategy to enhance the chemotherapy efficacy in AML.
  4. Blood. 2022 Mar 14. pii: blood.2021014520. [Epub ahead of print]
      Older patients with acute myeloid leukemia (AML) have high relapse risk and poor survival after allogeneic hematopoietic cell transplantation (HCT). Younger patients may receive myeloablative conditioning to mitigate relapse risk associated with high-risk genetics or measurable residual disease (MRD), but older adults typically receive reduced-intensity conditioning (RIC) to limit toxicity. To identify factors that drive HCT outcomes in older patients, we performed targeted mutational analysis (VAF≥2%) on diagnostic samples from 295 AML patients age 60 or older who underwent HCT in first complete remission, 91% of whom received RIC, and targeted duplex sequencing at the time of remission in 192 patients. In a multivariable model for leukemia-free survival (LFS) including baseline genetic and clinical variables, we defined patients with low (3-year LFS 85%), intermediate (55%), high (35%), and very high risk (7%). Prior to HCT, 79.7% of patients had persistent baseline mutations, including 18.3% with only DNMT3A or TET2 mutations (DT) and 61.4% with other mutations (MRDpositive). In univariable analysis, MRD-positivity was associated with increased relapse and inferior LFS compared with DT and MRDnegative patients. However, in a multivariable model accounting for baseline risk, MRD-positivity had no independent impact on LFS, likely due to its significant association with diagnostic genetic characteristics including MDS-associated gene mutations, TP53 mutations, and high-risk karyotype. In conclusion, molecular associations with MRD positivity and transplant outcomes in older AML patients are driven primarily by baseline genetics, and not by mutations present in remission. In this group of patients, where high-intensity conditioning carries substantial risk of toxicity, alternative approaches to mitigating MRD-associated relapse risk are required.
  5. J Hematol Oncol. 2022 Mar 12. 15(1): 25
      Acute myeloid leukemia (AML) patients suffer dismal prognosis upon treatment resistance. To study functional heterogeneity of resistance, we generated serially transplantable patient-derived xenograft (PDX) models from one patient with AML and twelve clones thereof, each derived from a single stem cell, as proven by genetic barcoding. Transcriptome and exome sequencing segregated clones according to their origin from relapse one or two. Undetectable for sequencing, multiplex fluorochrome-guided competitive in vivo treatment trials identified a subset of relapse two clones as uniquely resistant to cytarabine treatment. Transcriptional and proteomic profiles obtained from resistant PDX clones and refractory AML patients defined a 16-gene score that was predictive of clinical outcome in a large independent patient cohort. Thus, we identified novel genes related to cytarabine resistance and provide proof of concept that intra-tumor heterogeneity reflects inter-tumor heterogeneity in AML.
    Keywords:  Genetic barcoding; Heterogeneity; In vivo treatment; Single cell; Therapy resistance; Xenograft mouse model
  6. Leuk Res. 2022 Mar 06. pii: S0145-2126(22)00047-9. [Epub ahead of print]115 106821
    PETHEMA group
      Treatment of acute myeloid leukemia (AML) evolving from myeloproliferative (MPN) or myelodysplastic/myeloproliferative neoplasms (MDS/MPN) is challenging. We evaluated disease characteristics, treatment patterns and outcomes in 372 patients diagnosed with AML after MPN or MDS/MPN over a 27-year period. Frontline treatment was intensive chemotherapy (38%), hypomethylating agents [HMAs] (17%), non-intensive chemotherapy (14%), and supportive care (31%). Median overall survival was 4.8 months, with a 5-year survival rate of 4%. Median survival was 2.8, 3.9 and 8.3 months for the 1992-2010, 2011-2015 and 2016-2019 periods, respectively (test for trend p < 0.001). Complete response (CR) rate was higher with intensive chemotherapy (43%) than with non-intensive chemotherapy (12%) or HMAs (8.5%) [p < 0.001], but responses were short-lived without allogeneic hematopoietic cell transplantation. Patients treated with intensive chemotherapy or HMAs had superior survival than those receiving non-intensive chemotherapy (median: 8.5 vs. 8.6 vs. 4.2 months, respectively). No differences in treatment response or survival were observed according to prior disease subtypes. Patients undergoing transplantation in CR had better survival than those transplanted in other response categories (3-year survival rate of 64% vs. 22%, p = 0.002). Our results support the use of intensive chemotherapy followed by transplant whenever possible, and the preferential use of HMAs over attenuated chemotherapy regimens in unfit patients. In spite of the survival improvement in recent years, this subset of AML constitutes an unmet medical need and deserves systematic incorporation in clinical trials.
    Keywords:  Acute leukemia; Myelodysplastic/myeloproliferative neoplasm; Myeloproliferative neoplasm; Survival; Treatment
  7. Leuk Lymphoma. 2022 Mar 15. 1-8
      Antifungal prophylaxis (AFP) is recommended for acute myeloid leukemia (AML) patients receiving the combination of venetoclax (VEN) and a hypomethylating agent (HMA), but the benefit of this practice is unclear. We identified 131 patients with newly diagnosed AML who received frontline VEN/HMA and evaluated the use of AFP and its association with invasive fungal infections (IFIs) and AML outcomes. Seventeen percent of our patients received AFP at any time. Overall incidence of any IFI ('possible,' 'probable,' or 'proven' infection, as defined by the European Mycoses Study Group) was 13%, and the incidence did not differ based on AFP use (p=.74). Median overall survival did not differ based on AFP use or lack thereof (8.1 vs. 12.5 months, respectively; p=.14). Our findings suggest that, at an institution where the incidence of fungal infections is low, there does not appear to be a role for AFP in newly diagnosed AML patients receiving VEN/HMA.
    Keywords:  Acute myeloid leukemia; antifungal; fungal infection; prophylaxis
  8. Blood Adv. 2022 Mar 17. pii: bloodadvances.2021005992. [Epub ahead of print]
      Lipid raft-associated proteins play a vital role in membrane-mediated processes. The lipid microdomain-associated protein flotillin 2 (FLOT2), which has scaffolding function, is involved in polarization, as well as in actin cytoskeletal organization of primitive and mature hematopoietic cells and has been associated with different malignancies. However, its involvement in myeloid leukemias is not well studied. Using murine transplantation models, we show here that absence of FLOT2 from leukemia- initiating cells (LIC) altered disease course of BCR-ABL1+ chronic myeloid leukemia (CML), but not of MLL-AF9-driven acute myeloid leukemia (AML). While FLOT2 was required for expression of the adhesion molecule CD44 on both CML- and AML-LIC, a defect in the cytoskeleton, cell polarity and impaired homing ability of LIC was only observed in FLOT2-deficient BCR-ABL1+ compared to MLL-AF9+ cells. Downstream of CD44, BCR-ABL1-kinase-independent discrepancies were observed regarding expression, localization and activity of cell division control protein 42 homolog (CDC42) between wildtype and FLOT2-deficient human CML and AML cells. Inhibition of CDC42 by ML141 impaired the homing of CML LIC and, thereby, CML progression. This suggested that alteration of both CD44 and CDC42 may be causative of impaired CML progression in absence of FLOT2. In summary, our data suggest a FLOT2-CD44-CDC42 axis, which differentially regulates CML versus AML progression, with deficiency of FLOT2 impairing the development of CML.
  9. Nat Commun. 2022 Mar 17. 13(1): 1434
      Myeloid neoplasms are clonal hematopoietic stem cell disorders driven by the sequential acquisition of recurrent genetic lesions. Truncating mutations in the chromatin remodeler ASXL1 (ASXL1MT) are associated with a high-risk disease phenotype with increased proliferation, epigenetic therapeutic resistance, and poor survival outcomes. We performed a multi-omics interrogation to define gene expression and chromatin remodeling associated with ASXL1MT in chronic myelomonocytic leukemia (CMML). ASXL1MT are associated with a loss of repressive histone methylation and increase in permissive histone methylation and acetylation in promoter regions. ASXL1MT are further associated with de novo accessibility of distal enhancers binding ETS transcription factors, targeting important leukemogenic driver genes. Chromatin remodeling of promoters and enhancers is strongly associated with gene expression and heterogenous among overexpressed genes. These results provide a comprehensive map of the transcriptome and chromatin landscape of ASXL1MT CMML, forming an important framework for the development of novel therapeutic strategies targeting oncogenic cis interactions.
  10. Leuk Res. 2022 Mar 09. pii: S0145-2126(22)00048-0. [Epub ahead of print]115 106822
      Mutations characterize diverse human cancers; there is a positive correlation between elevated mutation frequency and tumor progression. One exception is acute myeloid leukemia (AML), which has few clonal single nucleotide mutations. We used highly sensitive and accurate Duplex Sequencing (DS) to show now that AML, in addition, has an extensive repertoire of variants with low allele frequencies, < 1%, which is below the accurate detection limit of most other sequencing methodologies. The subclonal variants are unique to each individual and change in composition, frequency, and sequence context from diagnosis to relapse. Their functional significance is apparent by the observation that many are known variants and cluster within functionally important protein domains. Subclones provide a reservoir of variants that could expand and contribute to the development of drug resistance and relapse. In accord, we accurately identified subclonal variants in AML driver genes NRAS and RUNX1 at allele frequencies between 0.1% and 0.3% at diagnosis, which expanded to comprise a major fraction (14-53%) of the blast population at relapse. Early and accurate detection of subclonal variants with low allele frequency thus offers the opportunity for early intervention, prior to detection of clinical relapse, to improve disease outcome and enhance patient survival.
    Keywords:  Acute myeloid leukemia; Diagnosis; Duplex Sequencing; Early detection; Relapse; Subclonal variants
  11. Nat Cell Biol. 2022 Mar;24(3): 299-306
      Transfer RNA-derived fragments (tRFs) are emerging small noncoding RNAs that, although commonly altered in cancer, have poorly defined roles in tumorigenesis1. Here we show that pseudouridylation (Ψ) of a stem cell-enriched tRF subtype2, mini tRFs containing a 5' terminal oligoguanine (mTOG), selectively inhibits aberrant protein synthesis programmes, thereby promoting engraftment and differentiation of haematopoietic stem and progenitor cells (HSPCs) in patients with myelodysplastic syndrome (MDS). Building on evidence that mTOG-Ψ targets polyadenylate-binding protein cytoplasmic 1 (PABPC1), we employed isotope exchange proteomics to reveal critical interactions between mTOG and functional RNA-recognition motif (RRM) domains of PABPC1. Mechanistically, this hinders the recruitment of translational co-activator PABPC1-interacting protein 1 (PAIP1)3 and strongly represses the translation of transcripts sharing pyrimidine-enriched sequences (PES) at the 5' untranslated region (UTR), including 5' terminal oligopyrimidine tracts (TOP) that encode protein machinery components and are frequently altered in cancer4. Significantly, mTOG dysregulation leads to aberrantly increased translation of 5' PES messenger RNA (mRNA) in malignant MDS-HSPCs and is clinically associated with leukaemic transformation and reduced patient survival. These findings define a critical role for tRFs and Ψ in difficult-to-treat subsets of MDS characterized by high risk of progression to acute myeloid leukaemia (AML).
  12. Cancer Med. 2022 Mar 16.
      BACKGROUND: Acute myeloid leukemia (AML) patients have limited effect from T-cell-based therapies, such as PD-1 and CTLA-4 blockade. However, recent data indicate that AML patients with TP53 mutation have higher immune infiltration and other immunomodulatory therapies could thus potentially be effective. Here, we performed the transcriptional analysis of distinct T-cell subpopulations from TP53-mutated AML to identify gene expression signatures suggestive of altered functional properties.METHODS: CD8+ cytotoxic T lymphocytes (CTLs), conventional helper T cells (Th), and regulatory T cells (Tregs) were sorted from peripheral blood of AML patients with TP53 mutation (n = 5) and healthy donors (n = 3), using FACS, and the different subpopulations were subsequently subjected to RNA-sequencing. Differentially expressed genes were identified and gene set enrichment analysis (GSEA) was performed to outline altered pathways and exhaustion status. Also, expression levels for a set of genes encoding established and emerging immuno-oncological targets were defined.
    RESULTS: The results showed altered transcriptional profiles for each of the T-cell subpopulations from TP53-mutated AML as compared to control subjects. IFN-α and IFN-γ signaling were stronger in TP53-mutated AML for both CTLs and Tregs. Furthermore, in TP53-mutated AML as compared to healthy controls, Tregs showed gene expression signatures suggestive of metabolic adaptation to their environment, whereas CTLs exhibited features of exhaustion/dysfunction with a stronger expression of TIM3 as well as enrichment of a gene set related to exhaustion.
    CONCLUSIONS: The results provide insights on mechanisms underlying the inadequate immune response to leukemic cells in TP53-mutated AML and open up for further exploration toward novel treatment regimens for these patients.
    Keywords:   TP53 ; RNA-sequencing; T-cells; acute myeloid leukemia; immunotherapy
  13. Br J Haematol. 2022 Mar 17.
      In adult acute myeloid leukaemia (AML), immunophenotypic differences enable discrimination of leukaemic stem cells (LSCs) from healthy haematopoietic stem cells (HSCs). However, immunophenotypic stem cell characteristics are less explored in paediatric AML. Employing a 15-colour flow cytometry assay, we analysed the expression of eight aberrant surface markers together with BCL-2 on CD34+ CD38- bone marrow stem cells from 38 paediatric AML patients and seven non-leukaemic, age-matched controls. Furthermore, clonality was investigated by genetic analyses of sorted immunophenotypically abnormal stem cells from six patients. A total of 50 aberrant marker positive (non-HSC-like) subsets with 41 different immunophenotypic profiles were detected. CD123, CLEC12A, and IL1RAP were the most frequently expressed markers. IL1RAP, CD93, and CD25 expression were not restricted to stem cells harbouring leukaemia-associated mutations. Differential BCL-2 expression was found among defined cytogenetic subgroups. Interestingly, only immunophenotypically abnormal non-HSC-like subsets demonstrated BCL-2 overexpression. Collectively, we observed pronounced immunophenotypic heterogeneity within the stem cell compartment of paediatric AML patients. Additionally, certain aberrant markers used in adults seemed to be ineligible for detection of leukaemia-representing stem cells in paediatric patients implying that inference from adult studies must be done with caution.
    Keywords:  AML stem cells; BCL-2; CD123; CLEC12A; IL1RAP; immunophenotyping; paediatric AML
  14. Mol Cell. 2022 Mar 17. pii: S1097-2765(22)00163-0. [Epub ahead of print]82(6): 1107-1122.e7
      Splicing factor mutations are common among cancers, recently emerging as drivers of myeloid malignancies. U2AF1 carries hotspot mutations in its RNA-binding motifs; however, how they affect splicing and promote cancer remain unclear. The U2AF1/U2AF2 heterodimer is critical for 3' splice site (3'SS) definition. To specifically unmask changes in U2AF1 function in vivo, we developed a crosslinking and immunoprecipitation procedure that detects contacts between U2AF1 and the 3'SS AG at single-nucleotide resolution. Our data reveal that the U2AF1 S34F and Q157R mutants establish new 3'SS contacts at -3 and +1 nucleotides, respectively. These effects compromise U2AF2-RNA interactions, resulting predominantly in intron retention and exon exclusion. Integrating RNA binding, splicing, and turnover data, we predicted that U2AF1 mutations directly affect stress granule components, which was corroborated by single-cell RNA-seq. Remarkably, U2AF1-mutant cell lines and patient-derived MDS/AML blasts displayed a heightened stress granule response, pointing to a novel role for biomolecular condensates in adaptive oncogenic strategies.
    Keywords:  AML; MDS; RNA; RNA binding; RNA granules; U2AF1; freCLIP; splicing; stress granules; stress response
  15. Blood Adv. 2022 Mar 14. pii: bloodadvances.2021006835. [Epub ahead of print]
      Hematopoietic stem cell (HSC) expansion with small molecules emerged as a promising avenue to improve the outcome of HSC transplantation, a procedure representing the main curative option for poor prognosis hematological diseases. HSC expansion also appears as a valuable resource for stem cell genetic engineering for therapeutic purposes. Unfortunately, the immuno-phenotype of expanded HSCs remains to be fully defined as some of the previously described HSC markers are not compatible with current culture conditions. Our group identified EPCR and ITGA3 as reliable markers that, when combined with currently known markers such as CD34 and CD90, define long-term reconstituting hematopoietic stem cells (LT-HSCs) in expanded cultures. In this article, we identify CEACAM1 as a novel culture-compatible surface marker of ex vivo expanded LT-HSCs that further refines the immuno-phenotype of these cells. CEACAM1-expressing cells show clonogenic and differentiation properties that are reminiscent of LT-HSCs in vitro, as well as long-term, serial and multilineage engraftment capacity in vivo. Sorting CEACAM1 expressing cells from the CD34+CD45RA-CD90+EPCR+ITGA3+ subset allows LT-HSC purification with incomparable purity, highlighting the value of this new marker.
  16. Genes Dev. 2022 Mar 17.
      Acute myeloid leukemia with KMT2A (MLL) rearrangements is characterized by specific patterns of gene expression and enhancer architecture, implying unique core transcriptional regulatory circuitry. Here, we identified the transcription factors MEF2D and IRF8 as selective transcriptional dependencies of KMT2A-rearranged AML, where MEF2D displays partially redundant functions with its paralog, MEF2C. Rapid transcription factor degradation followed by measurements of genome-wide transcription rates and superresolution microscopy revealed that MEF2D and IRF8 form a distinct core regulatory module with a narrow direct transcriptional program that includes activation of the key oncogenes MYC, HOXA9, and BCL2. Our study illustrates a mechanism of context-specific transcriptional addiction whereby a specific AML subclass depends on a highly specialized core regulatory module to directly enforce expression of common leukemia oncogenes.
    Keywords:  IRF8; KMT2A-rearranged AML; MEF2D; transcriptional addiction
  17. Leukemia. 2022 Mar 12.
      Myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) are reported in up to 20% patients with blastic plasmacytoid dendritic cell neoplasm (BPDCN), where a shared clonal origin is shown in individual case studies. In this study, we performed targeted next generating sequencing on multiple bone marrow (BM), skin or sorted cells from 51 BPDCN patients (68.7 years,14.4-84.7), and detected mutations in BM hematopoietic cells in 65% (30/46) and BPDCN in 100% (27/27), both components showing similar high frequencies of TET2 (60% versus 58%) and ASXL1 (33% versus 40%). Of 24 patients with paired mutation data, 13(54%) had shared mutations, with TET2(77%), ASXL1(37%) and ZRSR2(22%) the most commonly shared, and NRAS the most gained mutation in BPDCN(9/24, 38%). Karyotypic abnormalities were detected in 19/29(66%) BPDCN but only in 1/49 BM hematopoietic cells, providing additional evidence of clonal evolution. BM clonal hematopoiesis (CH) was associated with an older age (p < 0.001), being confounding factors in multivariate analysis; whereas <10% BM BPDCN infiltrate and stem cell transplant were associated with favorable outcomes. This study is the first to report a high prevalence of BM CH in BPDCN patients beyond an associated diagnosis of MDS/CMML, and demonstrates a frequent clonal relationship in elderly, findings contributing to the understanding of BPDCN clonal origin.
  18. Blood Cancer Discov. 2022 Mar 15. pii: bloodcandisc.BCD-21-0039-E.2021. [Epub ahead of print]
      Current murine models of myeloproliferative neoplasms (MPN) cannot examine how MPN progress from a single bone marrow source to the entire hematopoietic system. Using transplantation of knock-in JAK2V617F hematopoietic cells into a single irradiated leg, we show development of Polycythemia Vera (PV) from a single anatomical site in immuno-competent mice. Barcode experiments reveal that grafted JAK2V617F stem/progenitor cells migrate from the irradiated leg to non-irradiated organs such as contralateral leg and spleen, which is strictly required for development of PV. Mutant cells colonizing the non-irradiated leg efficiently induce PV in non-conditioned recipient mice and contain JAK2V617F hematopoietic stem/progenitor cells that express high levels of carbonic anhydrase 1 (CA1), a peculiar feature also found in CD34+ cells from PV patients. Finally, genetic and pharmacologic inhibition of CA1 efficiently suppresses PV development and progression in mice and decreased PV patients erythroid progenitors strengthening CA1 as a potent therapeutic target for PV.
  19. Exp Hematol. 2022 Mar 11. pii: S0301-472X(22)00122-9. [Epub ahead of print]
      Acute myeloid leukemia (AML) is an aggressive blood malignancy characterized by the accumulation of immature blood cells that can severely impede the normal functions of the hematopoietic system. AML still has a poor 5-year survival rate of around 30% and efforts to develop novel targeted therapies have been met with challenges. Allogeneic hematopoietic stem cell transplantation represents a potentially curative treatment for many AML patients. Donor immune cells, namely T cells and NK cells can help eliminate residual leukemia cells through the beneficial graft-versus-leukemia (GVL) effect. Nevertheless, malignant cells can still escape allogeneic immune surveillance and lead to disease relapse. Recent studies have provided insights into AML-specific immune evasion mechanisms, many of which are driven by epigenetic changes. This presents epigenetic regulators as promising therapeutic targets for designing post-transplant maintenance therapies. Therefore, this review aims to summarize AML immune evasion mechanisms with a focus given to the allogeneic immune environment. We discuss the roles of epigenetic regulators in driving immune escape and propose targeted strategies for preventing leukemia relapse. We then discuss the diverse immunomodulatory effects of epigenetic inhibitors and their potentials to enhance the GVL effect. The current landscape of maintenance therapy trials with epigenetic inhibitors and their clinical prospects are also assessed.
  20. Exp Hematol. 2022 Mar 09. pii: S0301-472X(22)00120-5. [Epub ahead of print]
      Fanconi Anemia (FA) is an inherited disorder of DNA repair with hematologic manifestations that range from anemia to bone marrow failure (BMF) to acute myeloid leukemia (AML). In a murine model of FA (Fancc-/- mice), we found BMF was accelerated by repeated attempts to induce emergency (stress) granulopoiesis; the process for granulocyte production during the innate immune response. Fancc-/- mice exhibited an impaired granulocytosis response and died with profound anemia during repeated challenge. In the current study, we found erythropoiesis and serum erythropoietin decreased in Fancc-/- and wild type (Wt) mice as emergency granulopoiesis peaked. Serum erythropoietin returned to baseline during steady state resumption, and compensatory proliferation of erythroid progenitors was associated with DNA-damage and apoptosis in Fancc-/- mice, but not Wt mice. The erythropoietin receptor activates Janus kinase 2 (Jak2) and we found treatment of Fancc-/- mice with ruxolitinib (Jak1/2-inhibitor) decreased anemia, enhanced granulocytosis, delayed clonal progression and prolonged survival during repeated emergency granulopoiesis episodes. This was associated with a decrease in DNA damage and apoptosis in Fancc-/- erythroid progenitors during this process. Transcriptome analysis of these cells identified enhanced activity of pathways for metabolism of reactive oxygen species, and decreased apoptosis and autophagy related pathways, as major ruxolitinib-effects in Fancc-/- mice. In contrast, ruxolitinib primarily influenced pathways involved in proliferation and differentiation in Wt mice. Ruxolitinib is approved for treatment of myeloproliferative disorders and graft versus host disease, suggesting the possibility of translational use as a bone marrow protectant in FA.
    Keywords:  DNA-damage response; DNA-repair; erythropoiesis; granulopoiesis; inflammasome; innate immunity
  21. Sci Adv. 2022 Mar 18. 8(11): eabm7688
      Tissue stem cells temporally change intrinsic mechanisms to meet physiological demands. However, little is known whether and how stem cells rely on distinct extrinsic maintenance mechanisms over time. Here, we found that hematopoietic stem cells (HSCs) temporally transition to depend on thrombopoietin (TPO), a key extrinsic factor, from E16.5 onward in the developing liver. Deletion of Tpo reduced mTOR activity, induced differentiation gene expression, and preferentially depleted metabolically active HSCs. Ectopic activation of the JAK2 or MAPK pathway did not rescue HSCs in Tpo-/- mice. Enforced activation of the mTOR pathway by conditionally deleting Tsc1 significantly rescued HSCs and their gene expression in Tpo-/- mice. Lin28b intrinsically promoted mTOR activation in HSCs, and its expression diminished over time. Conditional deletion of Lin28b further reduced mTOR activity and strongly exacerbated HSC depletion in Tpo-/- mice. Therefore, HSCs temporally transition from intrinsic LIN28B-dependent to extrinsic TPO-dependent maintenance in the developing liver.
  22. Nat Med. 2022 Mar 14.
      Anti-CD19 chimeric antigen receptor (CAR) T cell therapy has led to unprecedented responses in patients with high-risk hematologic malignancies. However, up to 60% of patients still experience disease relapse and up to 80% of patients experience CAR-mediated toxicities, such as cytokine release syndrome or immune effector cell-associated neurotoxicity syndrome. We investigated the role of the intestinal microbiome on these outcomes in a multicenter study of patients with B cell lymphoma and leukemia. We found in a retrospective cohort (n = 228) that exposure to antibiotics, in particular piperacillin/tazobactam, meropenem and imipenem/cilastatin (P-I-M), in the 4 weeks before therapy was associated with worse survival and increased neurotoxicity. In stool samples from a prospective cohort of CAR T cell recipients (n = 48), the fecal microbiome was altered at baseline compared to healthy controls. Stool sample profiling by 16S ribosomal RNA and metagenomic shotgun sequencing revealed that clinical outcomes were associated with differences in specific bacterial taxa and metabolic pathways. Through both untargeted and hypothesis-driven analysis of 16S sequencing data, we identified species within the class Clostridia that were associated with day 100 complete response. We concluded that changes in the intestinal microbiome are associated with clinical outcomes after anti-CD19 CAR T cell therapy in patients with B cell malignancies.
  23. Cell Stem Cell. 2022 Mar 10. pii: S1934-5909(22)00061-3. [Epub ahead of print]
      Abnormal nuclear morphology is a hallmark of malignant cells widely used in cancer diagnosis. Pelger-Huët anomaly (PHA) is a common abnormality of neutrophil nuclear morphology of unknown molecular etiology in myeloid neoplasms (MNs). We show that loss of nuclear lamin B1 (LMNB1) encoded on chromosome 5q, which is frequently deleted in MNs, induces defects in nuclear morphology and human hematopoietic stem cell (HSC) function associated with malignancy. LMNB1 deficiency alters genome organization inducing in vitro and in vivo expansion of HSCs, myeloid-biased differentiation with impaired lymphoid commitment, and genome instability due to defective DNA damage repair. Nuclear dysmorphology of neutrophils in patients with MNs is associated with 5q deletions spanning the LMNB1 locus, and lamin B1 loss is both necessary and sufficient to cause PHA in normal and 5q-deleted neutrophils. LMNB1 loss thus causes acquired PHA and links abnormal nuclear morphology with HSCs and progenitor cell fate determination via genome organization.
    Keywords:  3D genome; 5q deletions; genome instability; hematopoietic stem and progenitor cells; lineage determination; myeloid neoplasms; neutrophils; nuclear lamins; nuclear morphology
  24. Blood. 2022 Mar 16. pii: blood.2021014308. [Epub ahead of print]
      Hemogen, also known as EDAG, is a hematopoietic tissue-specific gene that regulates the proliferation and differentiation of hematopoietic cells. However, the mechanism underlying hemogen function in erythropoiesis is unknown. We found that depletion of hemogen in human CD34+ erythroid progenitor cells and HUDEP2 cells significantly reduced the expression of genes associated with heme and hemoglobin synthesis, supporting a positive role of hemogen in erythroid maturation. In human K562 cells, hemogen antagonized the occupancy of co-repressors NuRD complex and facilitated LDB1 complex-mediated chromatin looping. Hemogen recruited SWI/SNF complex ATPase BRG1 as a co-activator to regulate nucleosome accessibility and H3K27ac enrichment for promoter and enhancer activity. To ask if hemogen/BRG1 cooperativity is conserved in mammalian systems, we generated hemogen KO/KI mice and investigated hemogen/BRG1 function in murine erythropoiesis. Loss of hemogen in E12.5-E16.5 fetal liver cells impeded erythroid differentiation through reducing the production of mature erythroblasts. ChIP-seq in WT and hemogen KO animal revealed BRG1 is largely dependent on hemogen to regulate chromatin accessibility at erythroid gene promoters and enhancers. In summary, hemogen/BRG1 interaction in mammals is essential for fetal erythroid maturation and hemoglobin production through its active role in promoter and enhancer activity and chromatin organization.
  25. Nat Commun. 2022 Mar 14. 13(1): 1327
      In adult mammalian bone marrow (BM), vascular endothelial cells and perivascular reticular cells control the function of haematopoietic stem and progenitor cells (HSPCs). During fetal development, the mechanisms regulating the de novo haematopoietic cell colonization of BM remain largely unknown. Here, we show that fetal and adult BM exhibit fundamental differences in cellular composition and molecular interactions by single cell RNA sequencing. While fetal femur is largely devoid of leptin receptor-expressing cells, arterial endothelial cells (AECs) provide Wnt ligand to control the initial HSPC expansion. Haematopoietic stem cells and c-Kit+ HSPCs are reduced when Wnt secretion by AECs is genetically blocked. We identify Wnt2 as AEC-derived signal that activates β-catenin-dependent proliferation of fetal HSPCs. Treatment of HSPCs with Wnt2 promotes their proliferation and improves engraftment after transplantation. Our work reveals a fundamental switch in the cellular organization and molecular regulation of BM niches in the embryonic and adult organism.