bims-tremyl Biomed News
on Therapy resistance biology in myeloid leukemia
Issue of 2021‒05‒09
thirty-one papers selected by
Paolo Gallipoli
Barts Cancer Institute, Queen Mary University of London

  1. Blood Adv. 2021 May 11. 5(9): 2412-2425
      Advances in cancer genomics have revealed genomic classes of acute myeloid leukemia (AML) characterized by class-defining mutations, such as chimeric fusion genes or in genes such as NPM1, MLL, and CEBPA. These class-defining mutations frequently synergize with internal tandem duplications in FLT3 (FLT3-ITDs) to drive leukemogenesis. However, ∼20% of FLT3-ITD-positive AMLs bare no class-defining mutations, and mechanisms of leukemic transformation in these cases are unknown. To identify pathways that drive FLT3-ITD mutant AML in the absence of class-defining mutations, we performed an insertional mutagenesis (IM) screening in Flt3-ITD mice, using Sleeping Beauty transposons. All mice developed acute leukemia (predominantly AML) after a median of 73 days. Analysis of transposon insertions in 38 samples from Flt3-ITD/IM leukemic mice identified recurrent integrations at 22 loci, including Setbp1 (20/38), Ets1 (11/38), Ash1l (8/38), Notch1 (8/38), Erg (7/38), and Runx1 (5/38). Insertions at Setbp1 led exclusively to AML and activated a transcriptional program similar, but not identical, to those of NPM1-mutant and MLL-rearranged AMLs. Guide RNA targeting of Setbp1 was highly detrimental to Flt3ITD/+/Setbp1IM+, but not to Flt3ITD/+/Npm1cA/+, AMLs. Also, analysis of RNA-sequencing data from hundreds of human AMLs revealed that SETBP1 expression is significantly higher in FLT3-ITD AMLs lacking class-defining mutations. These findings propose that SETBP1 overexpression collaborates with FLT3-ITD to drive a subtype of human AML. To identify genetic vulnerabilities of these AMLs, we performed genome-wide CRISPR-Cas9 screening in Flt3ITD/+/Setbp1IM+ AMLs and identified potential therapeutic targets, including Kdm1a, Brd3, Ezh2, and Hmgcr. Our study gives new insights into epigenetic pathways that can drive AMLs lacking class-defining mutations and proposes therapeutic approaches against such cases.
  2. Blood. 2021 May 04. pii: blood.2020008229. [Epub ahead of print]
      Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy for which there is an unmet need for novel treatment strategies. Here, we characterize the growth arrest and DNA damage-inducible gene gamma (GADD45g) as a novel tumor suppressor in AML. We show that GADD45g is preferentially silenced in AML, especially in AML with FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutations and mixed-lineage leukemia (MLL)-rearrangements, and reduced expression of GADD45g is correlated with poor prognosis in AML patients. Upregulation of GADD45g impairs homologous recombination (HR) DNA repair, leading to DNA damage accumulation, and dramatically induces apoptosis, differentiation, growth arrest and increases sensitivity of AML cells to chemotherapeutic drugs, without affecting normal cells. In addition, GADD45g is epigenetically silenced by histone deacetylation in AML, and its expression is further downregulated by oncogenes FLT3-ITD and MLL-AF9 in patients carrying these genetic abnormalities. Combination of histone deacetylase 1/2 inhibitor Romidepsin with FLT3 tyrosine kinase inhibitor AC220 or bromodomain inhibitor JQ1 exert synergistic anti-leukemic effects on FLT3-ITD+ and MLL-AF9+ AML, respectively, by dually activating GADD45g. These findings uncover hitherto unreported evidence for the selective anti-leukemia role of GADD45g and provide novel strategies for the treatment of FLT3-ITD+ and MLL-AF9+ AML.
  3. Cell Death Discov. 2021 May 03. 7(1): 90
      Acute myeloid leukemia (AML) is a clinically and genetically heterogeneous clonal disease associated with unmet medical needs. Paralleling the pathology of other cancers, AML tumorigenesis and propagation can be ascribed to dysregulated cellular processes, including apoptosis. This function and others are regulated by tumor suppressor P53, which plays a pivotal role in leukemogenesis. Opposing P53-mediated activities is the mouse double minute 2 homolog (MDM2), which promotes P53 degradation. Because the TP53 mutation rate is low, and MDM2 frequently overexpressed, in patients with leukemia, targeting the MDM2-P53 axis to restore P53 function has emerged as an attractive AML treatment strategy. APG-115 is a potent MDM2 inhibitor under clinical development for patients with solid tumors. In cellular cultures and animal models of AML, we demonstrate that APG-115 exerted substantial antileukemic activity, as either a single agent or when combined with standard-of-care (SOC) hypomethylating agents azacitidine (AZA) and decitabine (DAC), or the DNA-damaging agent cytarabine (Ara-C). By activating the P53/P21 pathway, APG-115 exhibited potent antiproliferative and apoptogenic activities, and induced cell cycle arrest, in TP53 wild-type AML lines. In vivo, APG-115 significantly reduced tumor burden and prolonged survival. Combinations of APG-115 with SOC treatments elicited synergistic antileukemic activity. To explain these effects, we propose that APG-115 and SOC agents augment AML cell killing by complementarily activating the P53/P21 pathway and upregulating DNA damage. These findings and the emerging mechanism of action afford a sound scientific rationale to evaluate APG-115 (with or without SOC therapies) in patients with AML.
  4. Oncogene. 2021 May 06.
      Transcription factor MYB has recently emerged as a promising drug target for the treatment of acute myeloid leukemia (AML). Here, we have characterized a group of natural sesquiterpene lactones (STLs), previously shown to suppress MYB activity, for their potential to decrease AML cell proliferation. Unlike what was initially thought, these compounds inhibit MYB indirectly via its cooperation partner C/EBPβ. C/EBPβ-inhibitory STLs affect the expression of a large number of MYB-regulated genes, suggesting that the cooperation of MYB and C/EBPβ broadly shapes the transcriptional program of AML cells. We show that expression of GFI1, a direct MYB target gene, is controlled cooperatively by MYB, C/EBPβ, and co-activator p300, and is down-regulated by C/EBPβ-inhibitory STLs, exemplifying that they target the activity of composite MYB-C/EBPβ-p300 transcriptional modules. Ectopic expression of GFI1, a zinc-finger protein that is required for the maintenance of hematopoietic stem and progenitor cells, partially abrogated STL-induced myelomonocytic differentiation, implicating GFI1 as a relevant target of C/EBPβ-inhibitory STLs. Overall, our data identify C/EBPβ as a pro-leukemogenic factor in AML and suggest that targeting of C/EBPβ may have therapeutic potential against AML.
  5. Oncogene. 2021 May 06.
      The t(8;21) fusion product, AML1/ETO, and hypoxia-inducible factor 1α (HIF1α) form a feed-forward transcription loop that cooperatively transactivates the DNA methyltransferase 3a gene promoter that leads to DNA hypermethylation and drives leukemia cell growth. Suppression of the RNA N6-methyladenosine (m6A)-reader enzyme YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) specifically compromises cancer stem cells in acute myeloid leukemia (AML) but promotes hematopoietic stem cell expansion without derailing normal hematopoiesis. However, the relevance of expression between AML1/ETO-HIF1α loop and YTHDF2, and its functional relationship with t(8;21) AML have not been documented. Here, we show that YTHDF2 is highly expressed in t(8;21) AML patients and associated with a higher risk of relapse and inferior relapse-free survival. Knockdown of YTHDF2 in leukemia cells causes an impaired cell proliferation rate in vitro and in mice. Mechanistically, HIF1α is able to bind to the hypoxia-response elements of the 5'-untranslated region of the YTHDF2 gene and promotes the transactivity of the YTHDF2 promoter. Knockdown and overexpression of either AML1/ETO or HIF1α resulted in decreased and increased YTHDF2 protein and mRNA expression in t(8;21) AML cells. In particular, knockdown of YTHDF2 resulted in increased global mRNA m6A levels in t(8;21) AML cells, accompanied by increased TNF receptor superfamily member 1b (TNFRSF1b) mRNA and protein expression levels. Last, we demonstrated that the m6A methylation and expression levels of the TNFRSF1b gene were both negatively correlated with HIF1α expression levels. In conclusion, YTHDF2 is a downstream target of the AML1/ETO-HIF1α loop and promotes cell proliferation probably by modulating the global m6A methylation in t(8;21) AML.
  6. Blood Adv. 2021 May 11. 5(9): 2350-2361
      In an effort to identify acute myeloid leukemia (AML)-restricted targets for therapeutic development in AML, we analyzed the transcriptomes of 2051 children and young adults with AML and compared the expression profile with normal marrow specimens. This analysis identified a large cohort of AML-restricted genes with high expression in AML, but low to no expression in normal hematopoiesis. Mesothelin (MSLN), a known therapeutic target in solid tumors, was shown to be highly overexpressed in 36% of the AML cohort (range, 5-1077.6 transcripts per million [TPM]) and virtually absent in normal marrow (range, 0.1-10.7 TPM). We verified MSLN transcript expression by quantitative reverse transcription polymerase chain reaction, confirmed cell surface protein expression on leukemic blasts by multidimensional flow cytometry, and demonstrated that MSLN expression was associated with promoter hypomethylation. MSLN was highly expressed in patients with KMT2A rearrangements (P < .001), core-binding factor fusions [inv(16)/t(16;16), P < .001; t(8;21), P < .001], and extramedullary disease (P = .001). We also demonstrated the presence of soluble MSLN in diagnostic serum specimens using an MSLN-directed enzyme-linked immunosorbent assay. In vitro and in vivo preclinical efficacy of the MSLN-directed antibody-drug conjugates (ADCs) anetumab ravtansine and anti-MSLN-DGN462 were evaluated in MSLN+ leukemia cell lines in vitro and in vivo, as well as in patient-derived xenografts. Treatment with ADCs resulted in potent target-dependent cytotoxicity in MSLN+ AML. In this study, we demonstrate that MSLN is expressed in a significant proportion of patients with AML and holds significant promise as a diagnostic and therapeutic target in AML, and that MSLN-directed therapeutic strategies, including ADCs, warrant further clinical investigation.
  7. Haematologica. 2021 05 06.
      Cytokines are key regulators of tumor immune surveillance by controlling immune cell activity. Here, we investigated whether interleukin 4 (IL4) has antileukemic activity via immune-mediated mechanisms in an in vivo murine model of acute myeloid leukemia driven by the MLL-AF9 fusion gene. Although IL4 strongly inhibited leukemia development in immunocompetent mice, the effect was diminished in immune-deficient recipient mice, demonstrating that the antileukemic effect of IL4 in vivo is dependent on the host immune system. Using flow cytometric analysis and immunohistochemistry, we revealed that the antileukemic effect of IL4 coincided with an expansion of F4/80+ macrophages in the bone marrow and spleen. To elucidate whether this macrophage expansion was responsible of the antileukemic effect, we depleted macrophages in vivo with clodronate liposomes. Macrophage depletion eliminated the antileukemic effect of IL4, showing that macrophages mediated the IL4-induced killing of leukemia cells. In addition, IL4 enhanced murine macrophage-mediated phagocytosis of leukemia cells in vitro. Global transcriptomic analysis of macrophages revealed an enrichment of signatures associated with alternatively activated macrophages and increased phagocytosis upon IL4 stimulation. Notably, IL4 concurrently induced Stat6-dependent upregulation of CD47 on leukemia cells, which suppressed macrophage activity. Consistent with this finding, combining CD47 blockade with IL4 stimulation enhanced macrophage-mediated phagocytosis of leukemia cells. Thus, IL4 has two counteracting roles in regulating phagocytosis in mice; enhancing macrophage-mediated killing of leukemia cells, but also inducing CD47 expression that protects target cells from excessive phagocytosis. Taken together, our data suggests that combined strategies that activate macrophages and block CD47 have therapeutic potential in AML.
  8. Cancers (Basel). 2021 Apr 29. pii: 2143. [Epub ahead of print]13(9):
      All-trans retinoic acid (ATRA) and valproic acid (VP) have been tried in the treatment of non-promyelocytic variants of acute myeloid leukemia (AML). Non-randomized studies suggest that the two drugs can stabilize AML and improve normal peripheral blood cell counts. In this context, we used a proteomic/phosphoproteomic strategy to investigate the in vivo effects of ATRA/VP on human AML cells. Before starting the combined treatment, AML responders showed increased levels of several proteins, especially those involved in neutrophil degranulation/differentiation, M phase regulation and the interconversion of nucleotide di- and triphosphates (i.e., DNA synthesis and binding). Several among the differentially regulated phosphorylation sites reflected differences in the regulation of RNA metabolism and apoptotic events at the same time point. These effects were mainly caused by increased cyclin dependent kinase 1 and 2 (CDK1/2), LIM domain kinase 1 and 2 (LIMK1/2), mitogen-activated protein kinase 7 (MAPK7) and protein kinase C delta (PRKCD) activity in responder cells. An extensive effect of in vivo treatment with ATRA/VP was the altered level and phosphorylation of proteins involved in the regulation of transcription/translation/RNA metabolism, especially in non-responders, but the regulation of cell metabolism, immune system and cytoskeletal functions were also affected. Our analysis of serial samples during the first week of treatment suggest that proteomic and phosphoproteomic profiling can be used for the early identification of responders to ATRA/VP-based treatment.
    Keywords:  acute myeloid leukemia; all-trans retinoic acid; chemosensitivity; histone deacetylase; mass spectrometry; phosphoproteomics; proteomics; treatment; valproic acid
  9. Leukemia. 2021 May 06.
      Aberrant DNA methylation plays a pivotal role in tumor development and progression. DNA hypomethylating agents (HMA) constitute a class of drugs which are able to reverse DNA methylation, thereby triggering the re-programming of tumor cells. The first-generation HMA azacitidine and decitabine have now been in standard clinical use for some time, offering a valuable alternative to previous treatments in acute myeloid leukemia and myelodysplastic syndromes, so far particularly in older, medically non-fit patients. However, the longer we use these drugs, the more we are confronted with the (almost inevitable) development of resistance. This review provides insights into the mode of action of HMA, mechanisms of resistance to this treatment, and strategies to overcome HMA resistance including next-generation HMA and HMA-based combination therapies.
  10. Cancers (Basel). 2021 Apr 30. pii: 2161. [Epub ahead of print]13(9):
      To better understand the molecular basis of resistance to azacitidine (AZA) therapy in myelodysplastic syndromes (MDS) and acute myeloid leukemia with myelodysplasia-related changes (AML-MRC), we performed RNA sequencing on pre-treatment CD34+ hematopoietic stem/progenitor cells (HSPCs) isolated from 25 MDS/AML-MRC patients of the discovery cohort (10 AZA responders (RD), six stable disease, nine progressive disease (PD) during AZA therapy) and from eight controls. Eleven MDS/AML-MRC samples were also available for analysis of selected metabolites, along with 17 additional samples from an independent validation cohort. Except for two patients, the others did not carry isocitrate dehydrogenase (IDH)1/2 mutations. Transcriptional landscapes of the patients' HSPCs were comparable to those published previously, including decreased signatures of active cell cycling and DNA damage response in PD compared to RD and controls. In addition, PD-derived HSPCs revealed repressed markers of the tricarboxylic acid cycle, with IDH2 among the top 50 downregulated genes in PD compared to RD. Decreased citrate plasma levels, downregulated expression of the (ATP)-citrate lyase and other transcriptional/metabolic networks indicate metabolism-driven histone modifications in PD HSPCs. Observed histone deacetylation is consistent with transcription-nonpermissive chromatin configuration and quiescence of PD HSPCs. This study highlights the complexity of the molecular network underlying response/resistance to hypomethylating agents.
    Keywords:  IDH2; azacitidine therapy; histone acetylation; metabolic signature; myelodysplastic syndromes
  11. Leuk Lymphoma. 2021 May 04. 1-15
      The BCR-ABL1 fusion gene, which causes aberrant kinase activity and uncontrolled cell proliferation, is the hallmark of chronic myeloid leukemia (CML). The development of tyrosine kinase inhibitors (TKI) that target the BCR-ABL oncoprotein has led to dramatic improvement in CML management. However, some challenges remain to be addressed in the TKI era, including patient stratification and the selection of frontline TKIs and CML progression. Additionally, with the emerging goal of treatment-free remission (TFR) in CML management, biomarkers that predict the outcomes of stopping TKI remain to be identified. Notably, recent reports have revealed the power of genome screening in understanding the role of genome aberrations other than BCR-ABL1 in CML pathogenesis. These studies have discovered the presence of disease-phase specific mutations and linked certain mutations to inferior responses to TKI treatment and CML progression. A personalized approach that incorporates genetic data in tailoring treatment strategies has been successfully implemented in acute leukemia, and it represents a promising approach for the management of high-risk CML patients. In this article, we will review current knowledge about the mutational profile in different phases of CML as well as patterns of mutational dynamics in patients having different outcomes. We highlight the effects of somatic mutations involving certain genes (e.g. epigenetic modifiers) on the outcomes of TKI treatment. We also discuss the potential value of incorporating genetic data in treatment decisions and the routine care of CML patients as a future direction for optimizing CML management.
    Keywords:  Chronic myeloid leukemia; blast phase; personalized medicine; risk stratification; somatic mutations
  12. Oncotarget. 2021 Apr 27. 12(9): 878-890
      Acute myeloid leukemia (AML) is characterized by the proliferation of immature myeloid blasts and a suppressed immune state. Interferons have been previously shown to aid in the clearance of AML cells. Type I interferons are produced primarily by plasmacytoid dendritic cells (pDCs). However, these cells exist in a quiescent state in AML. Because pDCs express TLR 7-9, we hypothesized that the TLR7/8 agonist R848 would be able to reprogram them toward a more active, IFN-producing phenotype. Consistent with this notion, we found that R848-treated pDCs from patients produced significantly elevated levels of IFNβ. In addition, they showed increased expression of the immune-stimulatory receptor CD40. We next tested whether IFNβ would influence antibody-mediated fratricide among AML cells, as our recent work showed that AML cells could undergo cell-to cell killing in the presence of the CD38 antibody daratumumab. We found that IFNβ treatment led to a significant, IRF9-dependent increase in CD38 expression and a subsequent increase in daratumumab-mediated cytotoxicity and decreased colony formation. These findings suggest that the tolerogenic phenotype of pDCs in AML can be reversed, and also demonstrate a possible means of enhancing endogenous Type I IFN production that would promote daratumumab-mediated clearance of AML cells.
    Keywords:  acute myeloid leukemia; fratricide; interferon-beta; plasmacytoid dendritic cells
  13. Cell Death Dis. 2021 May 07. 12(5): 456
      Chronic myelogenous leukemia (CML) is a clonal malignancy of hematopoietic stem cells featured with the fusion protein kinase BCR-ABL. To elicit the mechanism underlying BCR-ABL stability, we perform a screen against a panel of deubiquitinating enzymes (DUBs) and find that the ubiquitin-specific protease 7 (USP7) drastically stabilizes the BCR-ABL fusion protein. Further studies show that USP7 interacts with BCR-ABL and blocks its polyubiquitination and degradation. Moreover, USP7 knockdown triggers BCR-ABL degradation and suppresses its downstream signaling transduction. In line with this finding, genetic or chemical inhibition of USP7 leads to BCR-ABL protein degradation, suppresses BCR/ABL signaling, and induces CML cell apoptosis. Furthermore, we find the antimalarial artesunate (ART) significantly inhibits USP7/BCR-ABL interaction, thereby promoting BCR-ABL degradation and inducing CML cell death. This study thus identifies USP7 as a putative Dub of BCR-ABL and provides a rationale in targeting USP7/BCR-ABL for the treatment of CML.
  14. Clin Transl Oncol. 2021 May 04.
      The relapse rate for children with acute myeloid leukemia is nearly 40% despite aggressive chemotherapy and often stem cell transplant. We sought to understand how environment-induced signaling responses are associated with clinical response to treatment. We previously reported that patients whose AML cells showed low G-CSF-induced STAT3 activation had inferior event-free survival compared to patients with stronger STAT3 responses. Here, we expanded the paradigm to evaluate multiple signaling parameters induced by a more physiological stimulus. We measured STAT3, STAT5 and ERK1/2 responses to G-CSF and to stromal cell-conditioned medium for 113 patients enrolled on COG trials AAML03P1 and AAML0531. Low inducible STAT3 activity was independently associated with inferior event-free survival in multivariate analyses. For inducible STAT5 activity, those with the lowest and highest responses had inferior event-free survival, compared to patients with intermediate STAT5 responses. Using existing RNA-sequencing data, we compared gene expression profiles for patients with low inducible STAT3/5 activation with those for patients with higher inducible STAT3/5 signaling. Genes encoding hematopoietic factors and mitochondrial respiratory chain subunits were overexpressed in the low STAT3/5 response groups, implicating inflammatory and metabolic pathways as potential mechanisms of chemotherapy resistance. We validated the prognostic relevance of individual genes from the low STAT3/5 response signature in a large independent cohort of pediatric AML patients. These findings provide novel insights into interactions between AML cells and the microenvironment that are associated with treatment failure and could be targeted for therapeutic interventions.
    Keywords:  Bone marrow stroma; Inflammation; Microenvironment; Pediatric AML; STAT3; STAT5
  15. Leukemia. 2021 May 05.
      Acute myeloid leukemia (AML) is a heterogeneous disease linked to a broad spectrum of molecular alterations, and as such, long-term disease control requires multiple therapeutic approaches. Driven largely by an improved understanding and targeting of these molecular aberrations, AML treatment has rapidly evolved over the last 3-5 years. The stellar successes of immunotherapies that harness the power of T cells to treat solid tumors and an improved understanding of the immune systems of patients with hematologic malignancies have led to major efforts to develop immunotherapies for the treatment of patients with AML. Several immunotherapies that harness T cells against AML are in various stages of preclinical and clinical development. These include bispecific and dual antigen receptor-targeting antibodies (targeted to CD33, CD123, CLL-1, and others), chimeric antigen receptor (CAR) T-cell therapies, and T-cell immune checkpoint inhibitors (including those targeting PD-1, PD-L1, CTLA-4, and newer targets such as TIM3 and STING). The current and future directions of these T-cell-based immunotherapies in the treatment landscape of AML are discussed in this review.
  16. Nat Immunol. 2021 May 06.
      Continuous supply of immune cells throughout life relies on the delicate balance in the hematopoietic stem cell (HSC) pool between long-term maintenance and meeting the demands of both normal blood production and unexpected stress conditions. Here we identified distinct subsets of human long-term (LT)-HSCs that responded differently to regeneration-mediated stress: an immune checkpoint ligand CD112lo subset that exhibited a transient engraftment restraint (termed latency) before contributing to hematopoietic reconstitution and a primed CD112hi subset that responded rapidly. This functional heterogeneity and CD112 expression are regulated by INKA1 through direct interaction with PAK4 and SIRT1, inducing epigenetic changes and defining an alternative state of LT-HSC quiescence that serves to preserve self-renewal and regenerative capacity upon regeneration-mediated stress. Collectively, our data uncovered the molecular intricacies underlying HSC heterogeneity and self-renewal regulation and point to latency as an orchestrated physiological response that balances blood cell demands with preserving a stem cell reservoir.
  17. Cancers (Basel). 2021 Apr 29. pii: 2156. [Epub ahead of print]13(9):
      Minimal residual disease (MRD) is now a powerful surrogate marker to assess the response to chemotherapy in acute myeloid leukemia (AML). DNMT3A mutation has been associated with adverse outcomes. In this study, we aimed to investigate the impact of DNMT3A status on NPM1 MRD predictive value for survival in a retrospective cohort of AML patients aged over 60 years old treated intensively. A total of 138 patients treated for NPM1-mutated AML in two French institutions were analyzed retrospectively. DNMT3A status did not influence the probability of having a ≥ 4log MRD1 reduction after induction. Only 20.4% of FLT3-ITD patients reached ≥ 4log MRD1 reduction compared to 47.5% in FLT3wt cases. A 4log reduction of NPM1 MRD was associated with a better outcome, even in FLT3-ITD mutated patients, independent of the allelic ratio. DNMT3A negative patients who reached a 4log reduction had a superior outcome to those who did not (HR = 0.23; p < 0.001). However, postinduction NPM1 MRD1 reduction was not predictive of OS and LFS in DNMT3Amut patients. These results confirm that post-induction NPM1 MRD1 is a reliable tool to assess disease outcome in elderly AML patients. However, the presence of DNMT3A also identifies a subgroup of patients at high risk of relapse.
    Keywords:  DNMT3A; NPM1; acute myeloid leukemia; elderly; prognosis
  18. Blood Adv. 2021 May 11. 5(9): 2362-2374
      Human anucleate platelets cannot be directly modified using traditional genetic approaches. Instead, studies of platelet gene function depend on alternative models. Megakaryocytes (the nucleated precursor to platelets) are the nearest cell to platelets in origin, structure, and function. However, achieving consistent genetic modifications in primary megakaryocytes has been challenging, and the functional effects of induced gene deletions on human megakaryocytes for even well-characterized platelet genes (eg, ITGA2B) are unknown. Here we present a rapid and systematic approach to screen genes for platelet functions in CD34+ cell-derived megakaryocytes called CRIMSON (CRISPR-edited megakaryocytes for rapid screening of platelet gene functions). By using CRISPR/Cas9, we achieved efficient nonviral gene editing of a panel of platelet genes in megakaryocytes without compromising megakaryopoiesis. Gene editing induced loss of protein in up to 95% of cells for platelet function genes GP6, RASGRP2, and ITGA2B; for the immune receptor component B2M; and for COMMD7, which was previously associated with cardiovascular disease and platelet function. Gene deletions affected several select responses to platelet agonists in megakaryocytes in a manner largely consistent with those expected for platelets. Deletion of B2M did not significantly affect platelet-like responses, whereas deletion of ITGA2B abolished agonist-induced integrin activation and spreading on fibrinogen without affecting the translocation of P-selectin. Deletion of GP6 abrogated responses to collagen receptor agonists but not thrombin. Deletion of RASGRP2 impaired functional responses to adenosine 5'-diphosphate (ADP), thrombin, and collagen receptor agonists. Deletion of COMMD7 significantly impaired multiple responses to platelet agonists. Together, our data recommend CRIMSON for rapid evaluation of platelet gene phenotype associations.
  19. Blood. 2021 May 04. pii: blood.2021010958. [Epub ahead of print]
      The BCL6 co-repressor (BCOR) is a transcription factor involved in the control of embryogenesis, mesenchymal stem cells function, hematopoiesis and lymphoid development. Recurrent somatic clonal mutations of the BCOR gene and its homologue BCORL1 have been detected in several hematological malignancies and aplastic anemia. They are scattered across the whole gene length and mostly represent frameshifts (deletions, insertions), nonsense and missence mutations. These disruptive events lead to the loss of full-length BCOR protein and to the lack or low expression of a truncated form of the protein, both consistent with the tumor suppressor role of BCOR. BCOR and BCORL1 mutations are similar to those causing two rare X-linked diseases: the oculo-facio-cardio-dental (OFCD) and the Shukla-Vernon syndromes, respectively. Here, we focus on the structure and function of normal BCOR and BCORL1 in normal hematopoietic and lymphoid tissues and review the frequency and clinical significance of the mutations of these genes in malignant and non-malignant hematological diseases. Moreover, we discuss the importance of mouse models to better understand the role of Bcor loss, alone and combined with alterations of other genes (e.g. Dnmt3a and Tet2), in promoting hematological malignancies and in providing a useful platform for the development of new targeted therapies.
  20. Transplant Proc. 2021 May 04. pii: S0041-1345(21)00236-0. [Epub ahead of print]
      Isolated extramedullary relapse (iEMR) of acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is rare and has a dismal prognosis. Among 67 patients with AML after allo-HSCT, iEMR and bone marrow relapse occurred in 6% and 20.9%, respectively, with a median time to relapse of 11.5 and 6.5 months, respectively. Here, we presented 4 iEMR-AML cases. Common relapse locations occurred in the central nervous system, skin, and lymph nodes. We also report a rare case of cardiac iEMR that responded to chemoradiotherapy. Two cases responded to local/systemic treatments, which resulted in prolonged survival. Another case had iEMR in the presence of chronic graft-versus-host disease. Bone marrow relapse occurring after iEMR was typical and found in three-fourths of the cases. In conclusion, iEMR-AML occurrence after allo-HSCT is not rare in Thai patients. Its unpredictability and lack of graft-versus-leukemia effect highlight the importance of monitoring EMR carefully and promptly providing treatments once it is detected.
  21. Br J Haematol. 2021 May 06.
      Little data are available for the expression of immune checkpoint (IC) molecules within myelodysplastic syndrome (MDS). Here, we report increased PD-L1+ CD34+ CD38- and PD-L1+ CD34+ CD38+ stem cell frequencies within MDS patients compared to stem cell recipients in remission. Additionally, we observed exceedingly similar PD1+ and Tim-3+ T-cell frequencies between acute myeloid leukaemia (AML) and MDS samples that were elevated compared to patients in remission. Furthermore, we found highly dynamic Tim-3+ and PD1+ T-cell frequencies within serial samples of relapsing MDS with excess blasts (MDS-EB II) patients, correlating with further disease markers. These findings support the idea of a potential successful implementation of IC inhibitor treatment in suitable MDS patients.
    Keywords:  AML; MDS; disease monitoring; exhausted T cells; flow cytometry; progenitor cells
  22. PLoS One. 2021 ;16(5): e0251011
      OBJECTIVE: The clinical characteristics and therapeutic strategy in acute myeloid leukemia (AML) are influenced by patients' age. We evaluated the impact of age on remission induction therapy for AML.METHODS: We retrospectively analyzed 3,011 adult AML patients identified from a nationwide database between January 2007 and December 2011.
    RESULTS: Three hundred twenty-nine (10.9%) acute promyelocytic leukemia (APL) and 2,682 (89.1%) non-APL patients were analyzed. The median age was 51 years and 55% of patients were male. Six hundred twenty-three patients (21%) were at favorable risk, 1522 (51%) were at intermediate risk, and 743 (25%) were at poor risk. As the age increased, the proportion of those at favorable risk and who received induction chemotherapy decreased. After induction therapy, complete response (CR) was achieved in 81.5% (243/298) of APL and 62.4% (1,409/2,258) of non-APL patients; these rates decreased as the age increased, with an obvious decrement in those older than 60 years. The median overall survival of non-APL patients was 18.7 months, while that of APL patients was not reached, with a 75% five-year survival rate.
    CONCLUSIONS: Age impacts both the biology and clinical outcomes of AML patients. Further studies should confirm the role of induction remission chemotherapy by age group.
  23. J Hematol Oncol. 2021 May 03. 14(1): 74
      IDH1 and IDH2 mutations (IDH1/2Mut) are recognized as recurrent genetic alterations in acute myeloid leukemia (AML) and associated with both clinical impact and therapeutic opportunity due to the recent development of specific IDH1/2Mut inhibitors. In T-cell acute lymphoblastic leukemia (T-ALL), their incidence and prognostic implications remain poorly reported. Our targeted next-generation sequencing approach allowed comprehensive assessment of genotype across the entire IDH1 and IDH2 locus in 1085 consecutive unselected and newly diagnosed patients with T-ALL and identified 4% of, virtually exclusive (47 of 49 patients), IDH1/2Mut. Mutational patterns of IDH1/2Mut in T-ALL present some specific features compared to AML. Whereas IDH2R140Q mutation was frequent in T-ALL (25 of 51 mutations), the IDH2R172 AML hotspot was absent. IDH2 mutations were associated with older age, an immature phenotype, more frequent RAS gain-of-function mutations and epigenetic regulator loss-of-function alterations (DNMT3A and TET2). IDH2 mutations, contrary to IDH1 mutations, appeared to be an independent prognostic factor in multivariate analysis with the NOTCH1/FBXW7/RAS/PTEN classifier. IDH2Mut were significantly associated with a high cumulative incidence of relapse and very dismal outcome, suggesting that IDH2-mutated T-ALL cases should be identified at diagnosis in order to benefit from therapeutic intensification and/or specific IDH2 inhibitors.
    Keywords:  IDH1; IDH2; T-ALL
  24. Br J Haematol. 2021 May 05.
      Sialic acid-binding immunoglobulin-like lectin (Siglec)-15 has recently been identified as a critical tumour checkpoint, augmenting the expression and function of programmed death-ligand 1. We raised a monoclonal antibody, A9E8, specific for Siglec-15 using phage display. A9E8 stained myeloid leukaemia cell lines and peripheral cluster of differentiation (CD)33+ blasts and CD34+ leukaemia stem cells from patients with acute myeloid leukaemia (AML). By contrast, there was minimal expression on healthy donor leucocytes or CD34+ stem cells from non-AML donors, suggesting targeting Siglec-15 may have significant therapeutic advantages over its fellow Siglec CD33. After binding, A9E8 was rapidly internalised (half-life of 180 s) into K562 cells. Antibodies to Siglec-15 therefore hold therapeutic potential for AML treatment.
    Keywords:  Siglec-15; acute myeloid leukaemia; antibody; endocytosis; phage display
  25. Leuk Res. 2021 Apr 24. pii: S0145-2126(21)00085-0. [Epub ahead of print]106 106584
      While dysregulation of MYC has been implicated in acute myeloid leukemia (AML), the impact of MYC protein expression in AML is less well understood. We investigated the correlation of MYC protein expression by immunohistochemistry (MYC-IHC) with MYC abnormalities and prognosis in adult de novo AML. MYC-IHC in bone marrow of patients with untreated AML (n = 58) was assessed and scored as MYClow (0-40 % of blasts) or MYChigh (> 40 % of blasts). This was correlated with MYC abnormalities by fluorescence in situ hybridization (FISH) and prognosis in the context of cytogenetic risk stratification. Residual myeloid disease at the end of induction was assessed by flow cytometry. MYClow and MYChigh were detected in 24 (41 %) and 34 cases (59 %), respectively. Extra copies of MYC were present in 12 % of cases and were not correlated with level of MYC-IHC. No cases had MYC translocation or amplification. Compared to MYClow patients, MYChigh patients had a shorter overall survival in all cytogenetic risk groups (68 vs. 21 months, p = 0.006) and in the intermediate risk group (61 vs. 21 months, p = 0.046). MYChigh patients had a tendency towards detected residual disease at the end of induction in all cytogenetic risk and intermediate risk groups. Regardless of the underlying mechanisms of MYC dysregulation, high level of MYC protein is expressed in the majority of AML and correlated to worse prognosis. Further studies on MYC dysregulation in leukemogenesis and therapy targeting MYC aberration are warranted.
    Keywords:  Acute myeloid leukemia; Cytogenetics; MYC; Prognosis
  26. Biochim Biophys Acta Mol Cell Res. 2021 May 01. pii: S0167-4889(21)00101-4. [Epub ahead of print] 119047
      The BH-3 mimetic venetoclax overcomes apoptosis and therapy resistance caused by high expression of BCL2 or loss of BH3-only protein function. Although a promising therapy for hematologic malignancies, increased expression of anti-apoptotic MCL-1 or BCL-XL, as well as other resistance mechanisms prevent a durable response to venetoclax. Recent studies demonstrate that agents targeting epigenetic mechanisms such as DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, histone methyltransferase EZH2 inhibitors, or bromodomain reader protein inhibitors may disable oncogenic gene expression signatures responsible for venetoclax resistance. Combination therapies including venetoclax and epigenetic therapies are effective in preclinical models and the subject of many current clinical trials. Here we review epigenetic strategies to overcome venetoclax resistance mechanisms in hematologic malignancies.
    Keywords:  EZH2; JQ1; azacytidine; decitabine; venetoclax
  27. Stem Cell Reports. 2021 Apr 27. pii: S2213-6711(21)00197-1. [Epub ahead of print]
      Advances in the isolation and gene expression profiling of single hematopoietic stem cells (HSCs) have permitted in-depth resolution of their molecular program. However, long-term HSCs can only be isolated to near purity from adult mouse bone marrow, thereby precluding studies of their molecular program in different physiological states. Here, we describe a powerful 7-day HSC hibernation culture system that maintains HSCs as single cells in the absence of a physical niche. Single hibernating HSCs retain full functional potential compared with freshly isolated HSCs with respect to colony-forming capacity and transplantation into primary and secondary recipients. Comparison of hibernating HSC molecular profiles to their freshly isolated counterparts showed a striking degree of molecular similarity, further resolving the core molecular machinery of HSC self-renewal while also identifying key factors that are potentially dispensable for HSC function, including members of the AP1 complex (Jun, Fos, and Ncor2), Sult1a1 and Cish. Finally, we provide evidence that hibernating mouse HSCs can be transduced without compromising their self-renewal activity and demonstrate the applicability of hibernation cultures to human HSCs.
    Keywords:  cell cycle; hematopoietic stem cells; quiescence; single-cell RNA sequencing; single-cell assays; stem cell niche; transplantation
  28. Br J Haematol. 2021 May 07.
      Older patients with acute myeloid leukaemia (AML) account for nearly half of those with the disease. Because they are perceived to be unfit for, unwilling to receive, or unlikely to benefit from conventional chemotherapy they represent an important unmet need. Tosedostat is a selective oral aminopeptidase inhibitor, which in phase I/II trials showed acceptable toxicity and encouraging efficacy. We report the only randomised study of low-dose cytosine arabinoside (LDAC) combined with tosedostat (LDAC-T) versus LDAC in untreated older patients not suitable for intensive treatment. A total of 243 patients were randomised 1:1 as part of the 'Pick-a-Winner' LI-1 trial. There was a statistically non-significant increase in the complete remission (CR) rate with the addition of tosedostat, LDAC-T 19% versus LDAC 12% [odds ratio (OR) 0·61, 95% confidence interval (CI) 0·30-1·23; P = 0·17]. For overall response (CR+CR with incomplete recovery of counts), there was little evidence of a benefit to the addition of tosedostat (25% vs. 18%; OR 0·68, 95% CI 0·37-1·27; P = 0·22). However, overall survival (OS) showed no difference (2-year OS 16% vs. 12%, hazard ratio 0·97, 95% CI 0·73-1·28; P = 0·8). Exploratory analyses failed to identify any subgroup benefitting from tosedostat. Despite promising pre-clinical, early non-randomised clinical data with acceptable toxicity and an improvement in response, we did not find evidence that the addition of tosedostat to LDAC produced a survival benefit in this group of patients with AML. International Standard Randomised Controlled Trial Number: ISRCTN40571019.
    Keywords:  AML; acute leukaemia; chemotherapy; elderly
  29. J Hematol Oncol. 2021 May 03. 14(1): 76
      BACKGROUND: Allogeneic hematopoietic cell transplantation (allo-HCT) using a mismatched unrelated donor (MMUD) and cord blood transplantation (CBT) are valid alternatives for patients without a fully human leukocyte antigen (HLA)-matched donor. Here, we compared the allo-HCT outcomes of CBT versus single-allele-mismatched MMUD allo-HCT with post-transplant cyclophosphamide (PTCy) in acute myeloid leukemia.METHODS: Patients who underwent a first CBT without PTCy (N = 902) or allo-HCT from a (HLA 9/10) MMUD with PTCy (N = 280) were included in the study. A multivariate regression analysis was performed for the whole population. A matched-pair analysis was carried out by propensity score-based 1:1 matching of patients (177 pairs) with known cytogenetic risk.
    RESULTS: The incidence of grade II-IV and grade III-IV acute graft-versus-host disease (GVHD) at 6 months was 36% versus 32% (p = 0.07) and 15% versus 11% (p = 0.16) for CBT and MMUD cohorts, respectively. CBT was associated with a higher incidence of graft failure (11% vs. 4%, p < 0.01) and higher 2-year non-relapse mortality (NRM) (30% vs. 16%, p < 0.01) compared to MMUD. In the multivariate analysis, CBT was associated with a higher risk of, NRM (HR = 2.09, 95% CI 1.46-2.99, p < 0.0001), and relapse (HR = 1.35, 95% CI 1-1.83, p = 0.05), which resulted in worse leukemia-free survival (LFS) (HR = 1.68, 95% CI 1.34-2.12, p < 0.0001), overall survival (OS) (HR = 1.7, 95% CI 1.33-2.17, p < 0.0001), and GVHD-free, relapse-free survival (GRFS) (HR = 1.49, 95% CI 1.21-1.83, p < 0.0001) compared to MMUD. The risk of grade II-IV acute GVHD (p = 0.052) and chronic GVHD (p = 0.69) did not differ significantly between the cohorts. These results were confirmed in a matched-pair analysis.
    CONCLUSIONS: CBT was associated with lower LFS, OS, and GRFS due to higher NRM, compared to MMUD allo-HCT with PTCy. In the absence of a fully matched donor, 9/10 MMUD with PTCy may be preferred over CBT.
    Keywords:  Acute leukemia; Acute myeloid leukemia; Allogeneic hematopoietic cell transplantation; Bone marrow; Cord blood transplantation; Cord blood unit; Disease relapse; Graft-versus-host disease; Human leukocyte antigen; Mismatched donor; Peripheral blood stem cell; Post-transplant cyclophosphamide; Toxicity