bims-tremyl Biomed News
on Therapy resistance biology in myeloid leukemia
Issue of 2021‒09‒26
37 papers selected by
Paolo Gallipoli
Barts Cancer Institute, Queen Mary University of London

  1. Blood Adv. 2021 Sep 20. pii: bloodadvances.2021005018. [Epub ahead of print]
      Acute myeloid leukemia (AML) often presents as an oligoclonal disease whereby multiple genetically distinct subclones can co-exist within patients. Differences in signaling and drug sensitivity of such subclones complicates treatment and warrants tools to identify them and track disease progression. We previously identified over 50 AML-specific plasma membrane (PM) proteins and seven of these (CD82, CD97, FLT3, IL1RAP, TIM3, CD25 and CD123) were implemented in routine diagnostics in patients with AML (n=256) and MDS (n=33). We developed a pipeline termed CombiFlow in which expression data of multiple PM markers is merged, allowing a Principle Component-based analyses to identify distinctive marker expression profiles and to generate single cell tSNE landscapes to longitudinally track clonal evolution. Positivity for one or more of the markers after 2 courses of intensive chemotherapy predicted a shorter relapse-free survival supporting a role of these markers in measurable residual disease (MRD) detection. CombiFlow also allowed the tracking of clonal evolution in paired diagnosis and relapse samples (n=12). Extending the panel to 36 AML-specific markers further refined the CombiFlow pipeline. In conclusion, CombiFlow provides a valuable tool in the diagnosis, MRD detection, clonal tracking, and the understanding of clonal heterogeneity in AML.
  2. Nat Genet. 2021 Sep 23.
      Altered transcription is a cardinal feature of acute myeloid leukemia (AML); however, exactly how mutations synergize to remodel the epigenetic landscape and rewire three-dimensional DNA topology is unknown. Here, we apply an integrated genomic approach to a murine allelic series that models the two most common mutations in AML: Flt3-ITD and Npm1c. We then deconvolute the contribution of each mutation to alterations of the epigenetic landscape and genome organization, and infer how mutations synergize in the induction of AML. Our studies demonstrate that Flt3-ITD signals to chromatin to alter the epigenetic environment and synergizes with mutations in Npm1c to alter gene expression and drive leukemia induction. These analyses also allow the identification of long-range cis-regulatory circuits, including a previously unknown superenhancer of Hoxa locus, as well as larger and more detailed gene-regulatory networks, driven by transcription factors including PU.1 and IRF8, whose importance we demonstrate through perturbation of network members.
  3. Adv Biol Regul. 2021 Sep 17. pii: S2212-4926(21)00046-4. [Epub ahead of print]82 100830
      Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy, characterized by a heterogeneous genetic landscape and complex clonal evolution, with poor outcomes. Mutation at the internal tandem duplication of FLT3 (FLT3-ITD) is one of the most common somatic alterations in AML, associated with high relapse rates and poor survival due to the constitutive activation of the FLT3 receptor tyrosine kinase and its downstream effectors, such as PI3K signaling. Thus, aberrantly activated FLT3-kinase is regarded as an attractive target for therapy for this AML subtype, and a number of small molecule inhibitors of this kinase have been identified, some of which are approved for clinical practice. Nevertheless, acquired resistance to these molecules is often observed, leading to severe clinical outcomes. Therapeutic strategies to tackle resistance include combining FLT3 inhibitors with other antileukemic agents. Here, we report on the preclinical activity of the combination of the FLT3 inhibitor quizartinib with the dual PI3K/mTOR inhibitor PF-04691502 in FLT3-ITD cells. Briefly, we show that the association of these two molecules displays synergistic cytotoxicity in vitro in FLT3-ITD AML cells, triggering 90% cell death at nanomolar concentrations after 48 h.
    Keywords:  Acute myeloid leukemia (AML); Combination therapy; Dual PI3K/mTOR inhibitors; FLT3-ITD; PF-04691502; Quizartinib
  4. Blood. 2021 Jun 03. pii: blood.2020010483. [Epub ahead of print]
      While clonal hematopoiesis (CH) can precede the development of acute myeloid leukemia (AML), it can also persist after achieving remission. Long-term clonal dynamics and clinical implications of persistent CH are not well understood. Here, we studied the prevalence, dynamics and clinical implications of post-remission CH in 164 AML patients who attained complete remission after induction chemotherapies. Post-remission CH was identified in 79 (49%) patients. Post-remission CH persisted long-term in 91% of the trackable patients despite treatment with various types of consolidation and maintenance therapies. Post-remission CH was eradicated in 20 out of 21 (95%) patients who underwent allogeneic stem cell transplant. While patients with post-remission CH as a group had comparable hematopoiesis with those without it, patients with persistent TET2 mutations showed significant neutropenia long-term. Post-remission CH had little impact on relapse risk, non-relapse mortality, and incidence of atherosclerotic cardiovascular disease, although the clinical impact of post-CR CH was heterogeneous among different mutations. These data suggest that while residual clonal hematopoietic stem cells (HSCs) are generally resistant to consolidation and maintenance therapies, they retain the ability to maintain normal hematopoiesis and have little impact on clinical outcomes, although larger study is needed to dissect the gene-specific heterogeneity.
  5. Blood Adv. 2021 Sep 21. pii: bloodadvances.2021004965. [Epub ahead of print]
      Patients with inv(16)/CBFB-MYH11 AML are considered favorable risk, however, nearly one-third relapse despite intensive therapy. Despite efforts to define risk groups within this favorable risk cohort, CBFB-MYH11 AML patients continue to be treated as a uniform cohort. Through transcriptome sequencing of 186 patients with inv(16) AML, we demonstrate that fusion junction breakpoints (exon 5-exon 33 versus other) are highly associated with outcome. The presence of exon 17 KIT mutations provides additional prognostic significance. Additionally, we provide insights into the transcriptional landscapes that differentiate these distinct CBFB-MYH11 AML subtypes. Children's Oncology Group trials include CCG-2961 (registered at as NCT00002798), AAML03P1 (NCT00070174), AAML0531 (NCT00372593), and AAML1031 (NCT01371981).
  6. Blood Adv. 2021 Sep 24. pii: bloodadvances.2021005236. [Epub ahead of print]
      We have recently identified the G protein-coupled neuropeptide receptor Calcitonin receptor-like (CALCRL) as an independent prognostic biomarker and a therapeutic target in more than 1500 adult patients with acute myeloid leukemia (AML). Here, we confirmed CALCRL expression as a prognostic factor in a cohort of 284 pediatric patients with AML. High CALCRL expression was independently associated with event free survival (EFS) (hazard ratio [HR], 1.87; 95% confidence interval [CI], 1.36-2.57; P=0.0001), overall survival (OS) (HR, 1.55; 95% [CI], 1.06-2.27; P=0.025) and cumulative incidence of relapse (CIR) (HR, 2.10; 95% CI, 1.49-1.96; P<0.0001) when adjusting for age, white blood cell count and genetic risk. Despite its association with leukemia stem cell (LSC) signatures, CALCRL expression remained associated with all endpoints when compared to the LSC17 score. The strong association of CALCRL expression with the risk of relapse also in the pediatric population supports its role as novel age-independent master regulator of relapse-initiating drug-tolerant AML cells in humans.
  7. Blood Adv. 2021 Sep 23. pii: bloodadvances.2021005455. [Epub ahead of print]
      Monitoring of NPM1 mutant (mut) measurable residual disease (MRD) in acute myeloid leukemia (AML) has an established role in patients treated with intensive chemotherapy. The European LeukemiaNet has defined molecular persistence at low copy number (MP-LCN) as MRD transcript level <1-2% with <1-log change between any 2 positive samples collected after the end of treatment (EOT). As the clinical impact of MP-LCN is unknown, we sought to characterize outcomes in patients with persistent NPM1mut MRD after EOT and identify factors associated with disease progression. Consecutive patients with newly diagnosed NPM1mut AML who received at least 2 cycles of intensive chemotherapy were included if NPM1mut MRD positive in the bone marrow at the EOT and not transplanted in first complete remission. One hundred patients were followed for a median of 23.5 months; 42% remained free of progression at 1 year: either spontaneously achieving complete molecular remission (30%) or retaining low-level NPM1mut transcript (12% for ≥12 months and 9% at last follow up). Forty percent met the criteria for MP-LCN. Pre-emptive salvage therapy was found to significantly prolong relapse-free survival. Risk factors associated with disease progression were concurrent FLT3-ITD at diagnosis and suboptimal MRD response (NPM1mut reduction <4.4-log) at EOT.
  8. Blood Adv. 2021 Sep 23. pii: bloodadvances.2021004583. [Epub ahead of print]
      Acute myeloid leukemia (AML) remains a difficult disease to treat disease. In a phase 2 clinical trial in patients with relapsed/refractory AML, combining the hypomethylating agent, azacitidine, with the PD-1 checkpoint inhibitor, nivolumab, demonstrated encouraging response rates (33%), median event-free and overall survival, compared with a historical cohort of contemporary patients treated with azacitidine-based therapies, with an acceptable safety profile. Biomarkers of response are yet to be determined. In this study, we leveraged a multiplexed immune assay to assess the functional states of CD4+ and CD8+ cells at a single-cell level in pretherapy bone marrows in 16 patients with R/R AML treated with azacitidine/nivolumab. Effector CD4+ but not CD8+ cells had distinct polyfunctional groups and were associated with responses and better outcomes. Further evaluation of the polyfunctional strength index composition across cell types revealed that IFN-g and TNF-a were the major drivers of enhanced polyfunctionality index of pretherapy CD4+ subset, while Granzyme B, IFN-g, MIP-1b and TNF-a drove the non-significantly enhanced pretreatment PSI of CD8+ subset in the responders. Single cell polyfunctional assays were predictive of response in AML and may have a potential role as a biomarker in the wider sphere of immunotherapy.
  9. Cell Rep. 2021 Sep 21. pii: S2211-1247(21)01174-8. [Epub ahead of print]36(12): 109725
      Despite absent expression in normal hematopoiesis, the Forkhead factor FOXC1, a critical mesenchymal differentiation regulator, is highly expressed in ∼30% of HOXAhigh acute myeloid leukemia (AML) cases to confer blocked monocyte/macrophage differentiation. Through integrated proteomics and bioinformatics, we find that FOXC1 and RUNX1 interact through Forkhead and Runt domains, respectively, and co-occupy primed and active enhancers distributed close to differentiation genes. FOXC1 stabilizes association of RUNX1, HDAC1, and Groucho repressor TLE3 to limit enhancer activity: FOXC1 knockdown induces loss of repressor proteins, gain of CEBPA binding, enhancer acetylation, and upregulation of nearby genes, including KLF2. Furthermore, it triggers genome-wide redistribution of RUNX1, TLE3, and HDAC1 from enhancers to promoters, leading to repression of self-renewal genes, including MYC and MYB. Our studies highlight RUNX1 and CEBPA transcription factor swapping as a feature of leukemia cell differentiation and reveal that FOXC1 prevents this by stabilizing enhancer binding of a RUNX1/HDAC1/TLE3 transcription repressor complex to oncogenic effect.
    Keywords:  FOXC1; Groucho; RUNX1; TLE3; acute myeloid leukemia
  10. Blood. 2021 Sep 20. pii: blood.2021010721. [Epub ahead of print]
      Uproleselan (GMI-1271) is a novel E-selectin antagonist that disrupts cell survival pathways, enhances chemotherapy response, improves survival in mouse xenograft and syngeneic models, and decreases chemotherapy toxicity in vivo. A phase 1/2 study (NCT02306291) evaluated the safety, tolerability, and anti-leukemic activity of uproleselan (5-20 mg/kg) with MEC (mitoxantrone, etoposide, cytarabine) among patients with relapsed/refractory (R/R) acute myeloid leukemia (AML). Among the first 19 patients, no dose-limiting toxicities were observed. The recommended phase 2 dose (RP2D) was 10 mg/kg twice daily. An additional 47 patients with R/R AML were treated with uproleselan at the RP2D plus MEC. At the RP2D, the remission rate (complete response [CR]/complete response with incomplete count recovery [CRi]) was 41% (CR 35%) and the median overall survival (OS) was 8.8 months. In a separate cohort, 25 newly diagnosed patients aged ≥60 years received uproleselan at the RP2D plus cytarabine and idarubicin (7+3). In these front-line patients, the CR/CRi rate was 72% (CR 52%) and the median OS was 12.6 months. The addition of uproleselan was associated with low rates of oral mucositis. E‑selectin ligand expression on leukemic blasts was higher in patients with relapsed vs primary refractory AML, and with high-risk cytogenetics and secondary AML in newly diagnosed older patients. In the R/R cohort, E‑selectin expression above 10% was associated with a higher response rate and improved survival. The addition of uproleselan to chemotherapy was well tolerated with high remission rates, low-induction mortality, and low rates of mucositis, providing strong rationale for phase 3 randomized confirmatory studies.
  11. Blood Cancer J. 2021 Sep 21. 11(9): 157
      The BCL2-inhibitor, Venetoclax (VEN), has shown significant anti-leukemic efficacy in combination with the DNMT-inhibitor, Azacytidine (AZA). To explore the mechanisms underlying the selective sensitivity of mutant leukemia cells to VEN and AZA, we used cell-based isogenic models containing a common leukemia-associated mutation in the epigenetic regulator ASXL1. KBM5 cells with CRISPR/Cas9-mediated correction of the ASXL1G710X mutation showed reduced leukemic growth, increased myeloid differentiation, and decreased HOXA and BCL2 gene expression in vitro compared to uncorrected KBM5 cells. Increased expression of the anti-apoptotic gene, BCL2, was also observed in bone marrow CD34+ cells from ASXL1 mutant MDS patients compared to CD34+ cells from wild-type MDS cases. ATAC-sequencing demonstrated open chromatin at the BCL2 promoter in the ASXL1 mutant KBM5 cells. BH3 profiling demonstrated increased dependence of mutant cells on BCL2. Upon treatment with VEN, mutant cells demonstrated increased growth inhibition. In addition, genome-wide methylome analysis of primary MDS samples and isogenic cell lines demonstrated increased gene-body methylation in ASXL1 mutant cells, with consequently increased sensitivity to AZA. These data mechanistically link the common leukemia-associated mutation ASXL1 to enhanced sensitivity to VEN and AZA via epigenetic upregulation of BCL2 expression and widespread alterations in DNA methylation.
  12. Front Oncol. 2021 ;11 726637
      Acute leukemia of ambiguous lineage (ALAL) is a rare type of leukemia and represents an unmet clinical need. In fact, due to heterogeneity, substantial rarity and absence of clinical trials, there are no therapeutic guidelines available. We investigated the genetic basis of 10 cases of ALAL diagnosed at our centre from 2008 and 2020, through a targeted myeloid and lymphoid sequencing approach. We show that this rare group of acute leukemias is enriched in myeloid-gene mutations. In particular we found that RUNX1 mutations, which have been found double mutated in 40% of patients and tend to involve both alleles, are associated with an undifferentiated phenotype and with lineage ambiguity. Furthermore, because this feature is typical of acute myeloid leukemia with minimal differentiation, we believe that our data strengthen the idea that acute leukemia with ambiguous lineage, especially those with an undifferentiated phenotype, might be genetically more closer to acute myeloid leukemia rather than acute lymphoblastic leukemia. These data enrich the knowledge on the genetic basis of ALAL and could have clinical implications as an acute myeloid leukemia (AML) - oriented chemotherapeutic approach might be more appropriate.
    Keywords:  Runx1; acute leukemia of ambiguous lineage; acute undifferentiated leukaemia; double mutations; myeloid genes
  13. Blood Adv. 2021 Sep 23. pii: bloodadvances.2021005089. [Epub ahead of print]
      Development of normal blood cells is often suppressed in juvenile myelomonocytic leukemia (JMML), a myeloproliferative neoplasm (MPN) of childhood, causing complications and impacting therapeutic outcomes. However, the mechanism underlying this phenomenon remains uncharacterized. To address this question, we induced the most common mutation identified in JMML (Ptpn11E76K) specifically in the myeloid lineage with hematopoietic stem cells (HSCs) spared. These mice uniformly developed a JMML-like MPN. Importantly, HSCs in the same bone marrow (BM) microenvironment were aberrantly activated and differentiated at the expense of self-renewal. As a result, HSCs lost quiescence and became exhausted. A similar result was observed in wild-type (WT) donor HSCs when co-transplanted with Ptpn11E76K/+ BM cells into WT mice. Co-culture testing demonstrated that JMML/MPN cells robustly accelerated differentiation in mouse and human normal hematopoietic stem/progenitor cells. Cytokine profiling revealed that Ptpn11E76K/+ MPN cells produced excessive IL-1β, but not IL-6, TNF-α, IFN-γ, IL-1α, or other inflammatory cytokines. Depletion of the IL-1β receptor effectively restored HSC quiescence, normalized their pool size, and rescued them from exhaustion in Ptpn11E76K/+/IL-1R-/- double mutant mice. These findings suggest IL-1β signaling as a potential therapeutic target for preserving normal hematopoietic development in JMML.
  14. Blood Adv. 2021 Sep 22. pii: bloodadvances.2021004881. [Epub ahead of print]
      Little is known about whether risk classification at diagnosis predicts post-hematopoietic cell transplantation (HCT) outcomes for acute myeloid leukemia (AML) patients. We evaluated 8709 AML patients from the CIBMTR database and, after selection and manual curation of cytogenetics data, 3779 patients in CR1 were included in the final analysis: 2384 with intermediate-risk, 969 with adverse-risk, and 426 with KMT2A-rearranged disease. An adjusted multivariable analysis compared to intermediate-risk patients detected an increased risk of relapse for KMT2A-rearranged and adverse-risk patients (HR 1.27, p = 0.01 and HR 1.71, p < 0.001, respectively). Leukemia-free survival (LFS) was similar for KMT2A and adverse-risk patients (HR 1.26, p = 0.002 and HR 1.47, p < 0.001), as was overall survival (OS) (HR 1.32, p < 0.001 and HR 1.45, p < 0.001). No differences in outcome could be detected when patients were stratified by KMT2A fusion partner. This is the largest study conducted to date on post-HCT outcomes in AML using manually curated cytogenetics for risk stratification. Our work demonstrates that risk classification at diagnosis remains predictive of post-HCT outcomes in AML. It also highlights the critical need to develop novel treatment strategies for patients with KMT2A rearrangements and adverse-risk disease.
  15. Oncotarget. 2021 Sep 14. 12(19): 1878-1885
      Acute myeloid leukemia (AML) is the most common type of leukemia and has a 5-year survival rate of 25%. The standard-of-care for AML has not changed in the past few decades. Promising immunotherapy options are being developed for the treatment of AML; yet, these regimens require highly laborious and sophisticated techniques. We create nanoTCEs using liposomes conjugated to monoclonal antibodies to enable specific binding. We also recreate the bone marrow niche using our 3D culture system and use immunocompromised mice to enable use of human AML and T cells with nanoTCEs. We show that CD33 is ubiquitously present on AML cells. The CD33 nanoTCEs bind preferentially to AML cells compared to Isotype. We show that nanoTCEs effectively activate T cells and induce AML killing in vitro and in vivo. Our findings suggest that our nanoTCE technology is a novel and promising immuno-therapy for the treatment of AML and provides a basis for supplemental investigations for the validation of using nanoTCEs in larger animals and patients.
    Keywords:  3D tissue culture model; T cell engagers; acute myeloid leukemia; nanoparticles
  16. Int J Hematol. 2021 Sep 21.
      All-trans retinoic acid (ATRA)-based therapy for acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia (AML), is the most successful example of differentiation therapy. Although ATRA can induce differentiation in some non-APL AML cell lines and primary blasts, clinical results of adding ATRA to standard therapy in non-APL AML patients have been inconsistent, probably due to use of different regimens and lack of diagnostic tools for identifying which patients may be sensitive to ATRA. In this study, we exposed primary blasts obtained from non-APL AML patients to ATRA to test for differentiation potential in vitro. We observed increased expression of differentiation markers, indicating a response to ATRA, in four out of fifteen primary AML samples. Three samples in which CD11b increased in response to ATRA had an inversion of chromosome 16 as well as the CBFB-MYH11 fusion gene, and the fourth sample was from a patient with KMT2A-rearranged, therapy-related AML. In conclusion, we identified a subgroup of non-APL AML patients with inv(16) and CBFB-MYH11 as the most sensitive to ATRA-mediated differentiation in vitro, and our results can help identify patients who may benefit from ATRA treatment.
    Keywords:  ATRA1; Acute myeloid leukemia2; CBFB-MYH11 5; Differentiation3; Inversion of chromosome 164
  17. Oncol Lett. 2021 Nov;22(5): 745
      Alterations in RAS oncogenes have been implicated in various types of cancer, including acute myeloid leukemia (AML). Considering that currently, there are no targeted therapies for patients with RAS-mutated AML despite the poor outcomes, RAF may be a potential target for AML. In this study, we first analyzed the efficacy of different MAPK inhibitors in AML cell lines. We found that LY3009120, a pan-RAF inhibitor, significantly decreased cell survival in RAS-mutated AML cell lines. We then investigated the synergistic effects of LY3009120 with either cytarabine or azacitidine. We found that the combination of low-dose cytarabine and LY3009120 showed a synergistic effect in NRAS-mutated HL-60 cells and KRAS-mutated NB4 cells. This effect was caused by a decrease in proliferation, induction of apoptosis, and cell growth arrest through a decrease in phosphorylated MEK and ERK along with a cytotoxic response occurring specifically for the RAS mutation of the pan-RAF inhibitor LY3009120. In addition, we confirmed that combination treatment with low-dose cytarabine and LY3009120 led to an increase in apoptosis in primary AML cells. Our findings indicate that combination therapy with pan-RAF inhibitor LY3009120 and low-dose cytarabine may be a promising treatment strategy for RAS-mutated AML.
    Keywords:  RAS; acute myeloid leukemia; combined therapy; cytarabine; mitogen-activated protein kinase
  18. Cell Death Dis. 2021 Sep 24. 12(10): 870
      Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm caused by the presence of tyrosine kinase BCR-ABL1 fusion protein, which deregulate transcription and mRNA translation. Tyrosine kinase inhibitors (TKIs) are the first-choice treatment. However, resistance to TKIs remains a challenge to cure CML patients. Here, we reveal that the m6A methyltransferase complex METTL3/METTL14 is upregulated in CML patients and that is required for proliferation of primary CML cells and CML cell lines sensitive and resistant to the TKI imatinib. We demonstrate that depletion of METTL3 strongly impairs global translation efficiency. In particular, our data show that METTL3 is crucial for the expression of genes involved in ribosome biogenesis and translation. Specifically, we found that METTL3 directly regulates the level of PES1 protein identified as an oncogene in several tumors. We propose a model in which nuclear METTL3/METTL14 methyltransferase complex modified nascent transcripts whose translation is enhanced by cytoplasmic localization of METTL3, independently from its catalytic activity. In conclusion, our results point to METTL3 as a novel relevant oncogene in CML and as a promising therapeutic target for TKI resistant CML.
  19. J Clin Invest. 2021 Sep 21. pii: e141401. [Epub ahead of print]
      Somatic mutations in the spliceosome gene U2AF1 are common in patients with myelodysplastic syndromes. U2AF1 mutations that code for the most common amino acid substitutions are always heterozygous, and the retained wild-type allele is expressed, suggesting that mutant hematopoietic cells may require the residual wild-type allele to be viable. We show that hematopoiesis and RNA splicing in U2af1 heterozygous knock-out mice was similar to control mice, but that deletion of the wild-type allele in U2AF1(S34F) heterozygous mutant expressing hematopoietic cells (i.e., hemizygous mutant) was lethal. These results confirm that U2AF1 mutant hematopoietic cells are dependent on the expression of wild-type U2AF1 for survival in vivo and that U2AF1 is a haplo-essential cancer gene. Mutant U2AF1 (S34F) expressing cells were also more sensitive to reduced expression of wild-type U2AF1 than non-mutant cells. Furthermore, mice transplanted with leukemia cells expressing mutant U2AF1 had significantly reduced tumor burden and improved survival after the wild-type U2af1 allele was deleted compared to when it was not deleted. These results suggest that selectively targeting the wild-type U2AF1 allele in heterozygous mutant cells could induce cancer cell death and be a therapeutic strategy for patients harboring U2AF1 mutations.
    Keywords:  Cancer; Hematology; Leukemias; Oncology; RNA processing
  20. Cell Rep. 2021 Sep 21. pii: S2211-1247(21)01192-X. [Epub ahead of print]36(12): 109739
      Histone lysine methylation functions at the interface of the extracellular environment and intracellular gene expression. DOT1L is a versatile histone H3K79 methyltransferase with a prominent role in MLL-fusion leukemia, yet little is known about how DOT1L responds to extracellular stimuli. Here, we report that DOT1L protein stability is regulated by the extracellular glucose level through the hexosamine biosynthetic pathway (HBP). Mechanistically, DOT1L is O-GlcNAcylated at evolutionarily conserved S1511 in its C terminus. We identify UBE3C as a DOT1L E3 ubiquitin ligase promoting DOT1L degradation whose interaction with DOT1L is susceptible to O-GlcNAcylation. Consequently, HBP enhances H3K79 methylation and expression of critical DOT1L target genes such as HOXA9/MEIS1, promoting cell proliferation in MLL-fusion leukemia. Inhibiting HBP or O-GlcNAc transferase (OGT) increases cellular sensitivity to DOT1L inhibitor. Overall, our work uncovers O-GlcNAcylation and UBE3C as critical determinants of DOT1L protein abundance, revealing a mechanism by which glucose metabolism affects malignancy progression through histone methylation.
    Keywords:  DOT1L; MLL; O-GlcNAcylation; UBE3C; glucose; hexosamine biosynthesis pathway; histone; leukemia; methylation; ubiquitination
  21. F1000Res. 2021 ;10 204
      Background: Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by genetic and epigenetic aberrations that alter the differentiation capacity of myeloid progenitor cells. The transcription factor CEBPα is frequently mutated in AML patients leading to an increase in DNA methylation in many genomic locations. Previously, it has been shown that ecCEBPα (extra coding CEBP α) - a lncRNA transcribed in the same direction as CEBPα gene - regulates DNA methylation of CEBPα promoter in cis. Here, we hypothesize that ecCEBPα could participate in the regulation of DNA methylation in trans. Method: First, we retrieved the methylation profile of AML patients with mutated CEBPα locus from The Cancer Genome Atlas (TCGA). We then predicted the ecCEBPα secondary structure in order to check the potential of ecCEBPα to form triplexes around CpG loci and checked if triplex formation influenced CpG methylation, genome-wide. Results: Using DNA methylation profiles of AML patients with a mutated CEBPα locus, we show that ecCEBPα could interact with DNA by forming DNA:RNA triple helices and protect regions near its binding sites from global DNA methylation. Further analysis revealed that triplex-forming oligonucleotides in ecCEBPα are structurally unpaired supporting the DNA-binding potential of these regions. ecCEBPα triplexes supported with the RNA-chromatin co-localization data are located in the promoters of leukemia-linked transcriptional factors such as MLF2. Discussion: Overall, these results suggest a novel regulatory mechanism for ecCEBPα as a genome-wide epigenetic modulator through triple-helix formation which may provide a foundation for sequence-specific engineering of RNA for regulating methylation of specific genes.
    Keywords:  Acute myeloid leukemia; DNA methylation; Triplex; extra-coding CEBPα; long non coding RNA
  22. Leukemia. 2021 Sep 24.
      While the understanding of the genomic aberrations that underpin chronic and acute myeloid leukaemia (CML and AML) has allowed the development of therapies for these diseases, limitations remain. These become apparent when looking at the frequency of treatment resistance leading to disease relapse in leukaemia patients. Key questions regarding the fundamental biology of the leukaemic cells, such as their metabolic dependencies, are still unresolved. Even though a majority of leukaemic cells are killed during initial treatment, persistent leukaemic stem cells (LSCs) and therapy-resistant cells are still not eradicated with current treatments, due to various mechanisms that may contribute to therapy resistance, including cellular metabolic adaptations. In fact, recent studies have shown that LSCs and treatment-resistant cells are dependent on mitochondrial metabolism, hence rendering them sensitive to inhibition of mitochondrial oxidative phosphorylation (OXPHOS). As a result, rewired energy metabolism in leukaemic cells is now considered an attractive therapeutic target and the significance of this process is increasingly being recognised in various haematological malignancies. Therefore, identifying and targeting aberrant metabolism in drug-resistant leukaemic cells is an imperative and a relevant strategy for the development of new therapeutic options in leukaemia. In this review, we present a detailed overview of the most recent studies that present experimental evidence on how leukaemic cells can metabolically rewire, more specifically the importance of OXPHOS in LSCs and treatment-resistant cells, and the current drugs available to target this process. We highlight that uncovering specific energy metabolism dependencies will guide the identification of new and more targeted therapeutic strategies for myeloid leukaemia.
  23. Transplant Cell Ther. 2021 Sep 19. pii: S2666-6367(21)01227-6. [Epub ahead of print]
      BACKGROUND: Disease relapse remains the major cause of death among patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) who receive an allogeneic hematopoietic cell transplant (allo-HCT). Maintenance treatment with FLT3 inhibitors and hypomethylating agents (HMA) has been studied in various clinical trials with mixed results.OBJECTIVE: To synthesize the current evidence on the efficacy and safety of FLT3 inhibitors and HMA for maintenance therapy following allo-HCT in AML and MDS.
    METHODS: For this systematic review and meta-analysis Cochrane Library, Google Scholar, Ovid Medline, Ovid Embase, PubMed, Scopus, and Web of Science Core Collection were searched from inception to March 2021 for studies on maintenance therapies following allo-HCT in AML and MDS. Studies were excluded if they were reviews, commentaries, case series with <5 patients, or basic research articles, not published in English, not on post-allo-HCT maintenance with FLT3 inhibitors or HMA in AML or MDS, or if they were clinical trials without published results or duplicate publications from the same patient cohort. Studies with insufficient reporting of the primary endpoint (2-year overall survival [OS]) and studies using FLT3 inhibitors or HMA for pre-emptive treatment of imminent relapse based on positive measurable residual disease testing were excluded. Random-effects models were used to pool response rates for the primary outcome of 2-year OS. Hazard ratios (HR) for death and relapse were calculated for studies that included a control group. Rates of relapse-free survival (RFS), non-relapse mortality and acute and chronic graft-versus-host-disease (GVHD) were studied as secondary endpoints. Downs and Black checklist and risk of bias assessments were used to gauge the quality of individual studies. The study protocol has been registered on PROSPERO (CRD42020187298).
    RESULTS: Our search strategy identified 5559 studies. Twenty-one studies with a total of 809 patients were included in the meta-analysis. 2-year OS rates were 81.7% (95% confidence interval [CI]: 73.8-87.7%) and 65.7% (95% CI: 55.1-74.9%) among patients treated with FLT3 inhibitors and HMA, respectively. In sensitivity analyses restricted to studies that included a control group, maintenance therapy with FLT3 inhibitors (HR for death: 0.41; 95% CI: 0.26-0.62) or HMA (HR: 0.45; 95% CI: 0.31-0.66) appeared superior to no maintenance therapy. 2-year RFS rates were 79.8% (95% CI: 75.0-83.9%) and 62.4% (95% CI: 50.6-72.9%) among patients treated with FLT3 inhibitors and HMA, respectively. Rates of any grade acute and chronic GVHD were 33.1% (95% CI: 25.4-41.8%; grade 3/4: 16.5%) and 42.5% (95% CI: 26.3-60.4%) among FLT3 inhibitor and 42.7% (95% CI: 33.5-52.4%; grade 3/4: 8.1%) and 41.5% (95% CI: 32.0-51.6%) among HMA-treated patients, respectively.
    CONCLUSION: Maintenance therapy with either FLT3 inhibitors or HMA following allo-HCT, can lead to prolonged and improved OS and RFS with a favorable safety profile. Additional studies are needed to define the optimal duration of treatment, the role of measurable residual disease status, and transplant characteristics in patient selection.
    Keywords:  AML; Acute myeloid leukemia; Hypomethylating agent; Maintenance therapy; Sorafenib; Transplant
  24. Blood. 2021 Jun 10. pii: blood.2020006610. [Epub ahead of print]
      t(4;11) MLL-AF4 acute leukemia is one of the most aggressive malignancies in the infant and pediatric population, yet we have little information on the molecular mechanisms responsible for disease progression. This impairs the development of therapeutic regimens that can address the aggressive phenotype and lineage plasticity of MLL-AF4-driven leukemogenesis. This study highlights novel mechanisms of disease development by focusing on two microRNAs upregulated in leukemic blasts from primary patient samples: miR-130b and miR-128a. We show that miR-130b and miR-128a are downstream targets of MLL-AF4 and can individually drive the transition from a pre-leukemic stage to an acute leukemia in an entirely murine Mll-AF4 in vivo model. They are also required to maintain the disease phenotype. Interestingly, miR-130b overexpression led to a mixed/B-cell precursor/myeloid leukemia, propagated by the lymphoid-primed multipotent progenitor population, whereas miR-128a overexpression resulted in a pro-B acute lymphoblastic leukemia, maintained by a highly expanded Il7r+ckit+ blast population. Molecular and phenotypic changes induced by these two miRNAs fully recapitulate the human disease, including central nervous system infiltration and activation of an MLL-AF4 expression signature. Furthermore, we identified two downstream targets of these microRNAs, NR2F6 and SGMS1, which in extensive validation studies are confirmed as novel tumour suppressors of MLL-AF4+ leukemia. Our integrative approach thus provides a platform for the identification of essential co-drivers of MLL-rearranged leukemias, in which the pre-leukemia to leukemia transition and lineage plasticity can be dissected and new therapeutic approaches can be tested.
  25. Comput Syst Oncol. 2021 Sep;pii: e1026. [Epub ahead of print]1(3):
      Event-free and overall survival remain poor for patients with acute myeloid leukemia. Chemoresistant clones contributing to relapse arise from minimal residual disease (MRD) or newly-acquired mutations. However, the dynamics of clones comprising MRD is poorly understood. We developed a predictive stochastic model, based on a multitype age-dependent Markov branching process, to describe how random events in MRD contribute to the heterogeneity in treatment response. We employed training and validation sets of patients who underwent whole genome sequencing and for whom mutant clone frequencies at diagnosis and relapse were available. The disease evolution and treatment outcome are subject to stochastic fluctuations. Estimates of malignant clone growth rates, obtained by model fitting, are consistent with published data. Using the estimates from the training set, we developed a function linking MRD and time of relapse, with MRD inferred from the model fits to clone frequencies and other data. An independent validation set confirmed our model. In a third data set, we fitted the model to data at diagnosis and remission and predicted the time to relapse. As a conclusion, given bone marrow genome at diagnosis and MRD at or past remission, the model can predict time to relapse, and help guide treatment decisions to mitigate relapse.
    Keywords:  acute myeloid leukemia; clonal evolution; minimal residual disease
  26. Blood Adv. 2021 Sep 20. pii: bloodadvances.2020003737. [Epub ahead of print]
      Somatic mutations are rare in pediatric AML (pAML), indicating alternate strategies are needed to identify targetable dependencies. We performed the first enhancer mapping of 3 pAML in 22 patient samples. Generally, pAML samples were distinct from adult AML 4 samples, and MLL (KMT2A)-rearranged samples were also distinct from non-KMT2A-5 rearranged samples. Focusing specifically on super-enhancers (SEs), we identified SEs 6 associated with many known leukemia regulators. The retinoic acid receptor alpha 7 (RARA) gene was differentially regulated in our cohort, and a RARA associated SE was 8 detected in 64% our cohort across all cyto/molecular subtypes tested. RARA SE-positive 9 pAML cell lines and samples demonstrated high RARA mRNA levels. These samples 10 were specifically sensitive to the synthetic RARA agonist tamibarotene in vitro, with 11 slowed proliferation, apoptosis induction, differentiation, and upregulated retinoid target 12 gene expression, compared to RARA SE-negative samples. Tamibarotene prolonged 13 survival and suppressed the leukemia burden of a RARA SE-positive pAML patient-14 derived xenograft (PDX) mouse model compared to a RARA SE-negative PDX. Our work 15 demonstrates that examining chromatin regulation can identify new, druggable 16 dependencies in pAML and provides rationale for a pediatric tamibarotene trial in children 17 with RARA-high AML.
  27. Mol Cell. 2021 Sep 15. pii: S1097-2765(21)00713-9. [Epub ahead of print]
      Initiation is the rate-limiting step in translation, and its dysregulation is vital for carcinogenesis, including hematopoietic malignancy. Thus, discovery of novel translation initiation regulators may provide promising therapeutic targets. Here, combining Ribo-seq, mass spectrometry, and RNA-seq datasets, we discovered an oncomicropeptide, APPLE (a peptide located in ER), encoded by a non-coding RNA transcript in acute myeloid leukemia (AML). APPLE is overexpressed in various subtypes of AML and confers a poor prognosis. The micropeptide is enriched in ribosomes and regulates the initiation step to enhance translation and to maintain high rates of oncoprotein synthesis. Mechanically, APPLE promotes PABPC1-eIF4G interaction and facilitates mRNA circularization and eIF4F initiation complex assembly to support a specific pro-cancer translation program. Targeting APPLE exhibited broad anti-cancer effects in vitro and in vivo. This study not only reports a previously unknown function of micropeptides but also provides new opportunities for targeting the translation machinery in cancer cells.
    Keywords:  Micropeptide; PABPC1; cancer development; eIF4F complex assembly; eIF4G; leukemia; mRNA circularization; non-coding RNA (ncRNA); small open reading frame (ORF); translation initiation
  28. Leukemia. 2021 Sep 20.
      In the current classification of the World Health Organization (WHO), bone marrow mastocytosis (BMM) is a provisional variant of indolent systemic mastocytosis (ISM) defined by bone marrow involvement and absence of skin lesions. However, no additional diagnostic criteria for BMM have been proposed. Within the registry dataset of the European Competence Network on Mastocytosis, we compared characteristics and outcomes of 390 patients with BMM and 1175 patients with typical ISM. BMM patients were significantly older, predominantly male, had lower tryptase and lower burden of neoplastic mast cells, and displayed a higher frequency of allergic reactions, mainly triggered by Hymenoptera, than patients with typical ISM. The estimated 10-year progression-free survival of BMM and typical ISM was 95.9% and 92.6%, respectively. In BMM patients defined by WHO-based criteria, the presence of one B-Finding and tryptase level ≥125 ng/mL were identified as risk factors for progression in multivariate analyses. BMM patients without any of these risk factors were found to have better progression-free survival (p < 0.05) and better overall survival (p < 0.05) than other ISM patients. These data support the proposal to define BMM as a separate SM variant characterized by SM criteria, absence of skin lesions, absence of B-Findings, and tryptase levels <125 ng/mL.
  29. Acta Haematol. 2021 Sep 22. 1-7
      Mixed lineage leukemia (MLL) T10 is a relatively rare partner for the KMT2A lysine (K)-specific methyltransferase 2A gene. The common features and coexisting mutations of acute myeloid leukemia (AML) patients with KMT2A-MLLT10 remain unknown. In this study, 10 adult AML patients with KMT2A-MLLT10 fusions were picked up from 496 AML patients by using RT-polymerase chain reaction (PCR) and/or fluorescence in situ hybridization, and then screened for mutations in the 49 genes panel with next-generation sequencing and PCR, followed by direct Sanger sequencing. Of the 10 unique individuals identified, 6 were male and 4 were female (M:F ratio, 1.5:1) with ages ranging from 19 to 52 years (median 39.5 years). Most (90%, 9/10) patients with KMT2A-MLLT10 were accompanied by additional mutations. Twelve mutated genes were detected, averaging 2.1 mutations per patient (range, 0-4). The most frequently mutated gene was NRAS (n = 5). Clinical and laboratory data pointed to common features: French American British-M5 subtype (n = 7), a high rate of relapse, and biomarkers CD33 (n = 10), CD117 (n = 9), CD13 (n = 8), and CD64 (n = 8). Overall, most patients harbored at least one mutation. A high incidence of mutations affecting the RAS signaling pathway or RAS regulating components was found in 50% (5/10) patients. The overall survival is about 12.0 months. Allogeneic-hematopoietic stem cell transplantation trends to improve survival in selected patients.
    Keywords:  KMT2A-mixed-lineage leukemia T10; Leukemia; NRAS; Next-generation sequencing
  30. Biochim Biophys Acta Mol Basis Dis. 2021 Sep 15. pii: S0925-4439(21)00201-5. [Epub ahead of print] 166268
      OBJECTIVE: Treatment of acute myeloid leukemia (AML) remains a challenge. It is urgent to understand the microenvironment to improve therapy and prognosis.METHODS: Bioinformatics methods were used to analyze transcription expression profile of AML patient samples with complete clinical information from UCSC Xena TCGA-AML datasets and validate with GEO datasets. Western blot, qPCR, RNAi and CCK8 assay were used to assay the effect of GPX1 expression on AML cell viability and the expression of genes of interest.
    RESULTS: Our analyses revealed that highly expressed GPX1 in AML patients links to unfavorable prognosis. GPX1 expression was positively associated with not only fraction levels of myeloid-derived suppressor cells (MDSCs), monocytes and T cell exhaustion, the expression levels of MDSC markers, MDSC-promoting CCR2 and immune inhibitory checkpoints (TIM3/Gal-9, SIRPα and VISTA), but also negatively with low fraction levels of CD4+ and CD8+ T cells. Silencing GPX1 expression reduced AML cell viability and CCR2 expression. Moreover, GPX1-targetd kinases were PKC family, SRC family, SYK and PAK1, which promote AML progression and the resistance to therapy. Furthermore, Additionally, GPX1-associated prognostic signature (GPS) is an independent risk factor with high area under curve (AUC) values of receiver operating characteristic (ROC) curves. High risk group based on GPS enriched not only with endocytosis which transfers mitochondria to favor AML cell survival in response to chemotherapy, but also NOTCH,WNT and TLR signaling which promote therapy resistance.
    CONCLUSION: Our results revealed the significant involvement of GPX1 in AML immunosuppression via and provided a prognostic signature for AML patients.
    Keywords:  CCR2; GPX1; SIRPα/CD47; TIM3/Gal-9; VISTA; acute myeloid leukemia; immunosuppresion; myeloid derived suppressor cells; prognosis
  31. Blood Cancer J. 2021 Sep 24. 11(9): 159
      The present study evaluated outcomes and prognostic factors in adult patients with acute myeloid leukemia (AML) after syngeneic hematopoietic stem cell transplantation (HSCT). Among patients in first complete remission (CR1), outcomes of syngeneic HSCT (Syn) were compared with those of autologous HSCT (Auto), allogeneic HSCT from human leukocyte antigen (HLA)-matched sibling donor (MSD), or allogeneic HSCT from HLA-matched unrelated donor (MUD). Among 11,866 patients receiving first HSCT, 26 in the Syn group were analyzed. The 5-year overall survival (OS) rate, the cumulative incidence of relapse, and the cumulative incidence of non-relapse mortality (NRM) were 47.8%, 59.6%, and 4.6%, respectively. The OS was significantly better in patients in CR1 (n = 13) than in patients in non-CR1 (P = 0.012). Furthermore, 39 patients in CR1 each were assigned to the Auto, MSD, and MUD groups using propensity score matching. The 5-year OS in the Syn (68.4%) was not significantly different from those in the Auto (55.9%, P = 0.265), MSD (62.4%, P = 0.419), or MUD (63.7%, P = 0.409) groups. A higher relapse in the Syn than in the MSD and MUD groups was offset by lower NRM. In summary, syngeneic HSCT might be an alternative option for AML patients in CR1.
  32. Br J Haematol. 2021 Sep 25.
      We assessed the diagnostic performances of erythropoietin and JAK2 mutations in 1,090 patients with suspected polycythemia who were referred for red cell mass (RCM) measurement. In patients with a high haematocrit and/or haemoglobin level, a low erythropoietin level (<=3·3 mUI/ml) and JAK2 mutation showed comparable positive predictive value (PPV) for true polycythemia (RCM>=125%), 92·1% and 90% respectively. A very-low erythropoietin level (<=1·99 mUI/ml) had a PPV of 100% for polycythemia vera (PV) diagnosis. We confirmed the correlations between RCM, erythropoietin and JAK2 variant allelic frequency in PV patients. This study prompts the need to revisit the role of EPO in PV diagnostic criteria.
    Keywords:  JAK2; erythropoietin; myeloproliferative neoplasms (MPN); polycythemia; red cell mass
  33. Bone Marrow Transplant. 2021 Sep 21.
      Haploidentical hematopoietic-cell transplantation using post-transplant cyclophosphamide(Haplo-PTCy) is a feasible procedure in children with haematologic malignancies. However, data of a large series of children with acute leukaemia(AL) in this setting is missing. We analysed 144 AL Haplo-PTCy paediatric recipients; median age was 10 years. Patients had acute lymphoblastic(ALL; n = 86) or myeloblastic leukaemia(AML; n = 58) and were transplanted in remission(CR1: n = 40; CR2: n = 57; CR3+: n = 27) or relapse (n = 20). Bone marrow was the graft source in 57%; donors were father (54%), mother (35%), or sibling (11%). Myeloablative conditioning was used in 87%. Median follow-up was 31 months. At day +100, cumulative incidence (CI) of neutrophil recovery and acute GVHD (II-IV) were 94% and 40%, respectively. At 2-years, CI of chronic GVHD and relapse, were 31%, 40%, and estimated 2-year overall survival (OS), leukaemia-free survival (LFS) and graft-versus-host-relapse-free survival (GRFS) were 52%, 44% and 34% respectively. For patients transplanted in remission, positive measurable residual disease (MRD) prior to transplant was associated with decreased LFS (p = 0.05) and GRFS (p = 0.003) and increased risk of relapse (p = 0.02). Mother donor was associated with increased risk of chronic GVHD (p = 0.001), decreased OS (p = 0.03) and GRFS (p = 0.004). Use of PBSC was associated with increased risk of chronic GVHD (p = 0.04). In conclusion, achieving MRD negativity pre-transplant, avoiding use of mother donors and PBSC as graft source may improve outcomes of Haplo-PTCy in children with AL.
  34. Haematologica. 2021 Sep 23.
      Thrombocytopenia is common in patients with myelofibrosis and is a well-established adverse prognostic factor. Both of the approved Janus kinase (JAK) inhibitors, ruxolitinib and fedratinib, can worsen thrombocytopenia and have not been evaluated in patients with severe thrombocytopenia (.
  35. J Med Chem. 2021 Sep 22.
      Secondary mutations of FLT3 have become the main mechanism of FLT3 inhibitor resistance that presents a significant clinical challenge. Herein, a series of pyrazole-3-amine derivatives were synthesized and optimized to overcome the common secondary resistance mutations of FLT3. The structure-activity relationship and molecular dynamics simulation studies illustrated that the ribose region of FLT3 could be occupied to help address the obstacle of secondary mutations. Among those derivatives, compound 67 exhibited potent and selective inhibitory activities against FLT3-ITD-positive acute myeloid leukemia (AML) cells and possessed equivalent potency against transformed BaF3 cells with a variety of secondary mutations. Besides, cellular mechanism assays demonstrated that 67 strongly inhibited phosphorylation of FLT3 and its downstream signaling factors, as well as induced cell cycle arrest and apoptosis in MV4-11 cells. In the MV4-11 xenograft models, 67 exhibited potent antitumor potency without obvious toxicity. Taken together, these results demonstrated that 67 might be a drug candidate for the treatment of FLT3-ITD-positive AML.
  36. Cell Stem Cell. 2021 Sep 15. pii: S1934-5909(21)00351-9. [Epub ahead of print]
      Extracellular vesicles (EVs) transfer complex biologic material between cells. However, the role of this process in vivo is poorly defined. Here, we demonstrate that osteoblastic cells in the bone marrow (BM) niche elaborate extracellular vesicles that are taken up by hematopoietic progenitor cells in vivo. Genotoxic or infectious stress rapidly increased stromal-derived extracellular vesicle transfer to granulocyte-monocyte progenitors. The extracellular vesicles contained processed tRNAs (tiRNAs) known to modulate protein translation. 5'-ti-Pro-CGG-1 was preferentially abundant in osteoblast-derived extracellular vesicles and, when transferred to granulocyte-monocyte progenitors, increased protein translation, cell proliferation, and myeloid differentiation. Upregulating EV transfer improved hematopoietic recovery from genotoxic injury and survival from fungal sepsis. Therefore, EV-mediated tiRNA transfer provides a stress-modulated signaling axis in the BM niche distinct from conventional cytokine-driven stress responses.
    Keywords:  bone marrow; extracellular vesicles; hematopoiesis; myeloid progenitors; niche; protein translation; signaling; tiRNAs