bims-tremyl Biomed News
on Therapy resistance biology in myeloid leukemia
Issue of 2021‒09‒12
thirty-four papers selected by
Paolo Gallipoli
Barts Cancer Institute, Queen Mary University of London

  1. Front Oncol. 2021 ;11 709543
      Loss-of-function mutations in the DNA demethylase TET2 are associated with the dysregulation of hematopoietic stem cell differentiation and arise in approximately 10% of de novo acute myeloid leukemia (AML). TET2 mutations coexist with other mutations in AML, including TP53 mutations, which can indicate a particularly poor prognosis. Ascorbate can function as an epigenetic therapeutic in pathological contexts involving heterozygous TET2 mutations by restoring TET2 activity. How this response is affected when myeloid leukemia cells harbor mutations in both TET2 and TP53 is unknown. Therefore, we examined the effects of ascorbate on the SKM-1 AML cell line that has mutated TET2 and TP53. Sustained treatment with ascorbate inhibited proliferation and promoted the differentiation of these cells. Furthermore, ascorbate treatment significantly increased 5-hydroxymethylcytosine, suggesting increased TET activity as the likely mechanism. We also investigated whether ascorbate affected the cytotoxicity of Prima-1Met, a drug that reactivates some p53 mutants and is currently in clinical trials for AML. We found that the addition of ascorbate had a minimal effect on Prima-1Met-induced cytotoxicity, with small increases or decreases in cytotoxicity being observed depending on the timing of treatment. Collectively, these data suggest that ascorbate could exert a beneficial anti-proliferative effect on AML cells harboring both TET2 and TP53 mutations whilst not interfering with targeted cytotoxic therapies such as Prima-1Met.
    Keywords:  APR-246; Prima-1Met; TET2; ascorbate; differentiation; epigenetic therapy; leukemia; vitamin C
  2. Front Oncol. 2021 ;11 686765
      Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with a high mortality rate and relapse risk. Although progress on the genetic and molecular understanding of this disease has been made, the standard of care has changed minimally for the past 40 years and the five-year survival rate remains poor, warranting new treatment strategies. Here, we applied a two-step screening platform consisting of a primary cell viability screening and a secondary metabolomics-based phenotypic screening to find synergistic drug combinations to treat AML. A novel synergy between the oxidative phosphorylation inhibitor IACS-010759 and the FMS-like tyrosine kinase 3 (FLT3) inhibitor AC220 (quizartinib) was discovered in AML and then validated by ATP bioluminescence and apoptosis assays. In-depth stable isotope tracer metabolic flux analysis revealed that IACS-010759 and AC220 synergistically reduced glucose and glutamine enrichment in glycolysis and the TCA cycle, leading to impaired energy production and de novo nucleotide biosynthesis. In summary, we identified a novel drug combination, AC220 and IACS-010759, which synergistically inhibits cell growth in AML cells due to a major disruption of cell metabolism, regardless of FLT3 mutation status.
    Keywords:  FLT3-inhibitor; acute myeloid leukemia; complex I inhibitor; high-throughput screening; metabolomics
  3. Blood Adv. 2021 Sep 10. pii: bloodadvances.2020003661. [Epub ahead of print]
      Acute myeloid leukemia (AML) cells are highly dependent on oxidative phosphorylation (OxPhos) for survival and continually adapt to fluctuations in nutrient and oxygen availability in the bone marrow (BM) microenvironment. We investigated how the BM microenvironment affects the response to OxPhos inhibition in AML by using a novel complex I OxPhos inhibitor, IACS-010759. Cellular adhesion, growth, and apoptosis assays, along with measurements of mtDNA expression and mitochondrial reactive oxygen species generation, indicated that direct interactions with BM stromal cells triggered compensatory activation of mitochondrial respiration and resistance to OxPhos inhibition in AML cells. Mechanistically, OxPhos inhibition induced (1) transfer of mesenchymal stem cell (MSC)-derived mitochondria to AML cells via tunneling nanotubes under direct-contact coculture conditions, and (2) mitochondrial fission with an increase in functional mitochondria and mitophagy in AML cells. Mitochondrial fission is known to enhance cell migration, and we observed mitochondrial transport to the leading edge of protrusions of migrating AML cells toward MSCs by electron microscopy analysis. We further demonstrated that cytarabine, a commonly used antileukemia agent, increased OxPhos inhibition-triggered mitochondrial transfer from MSCs to AML cells. Our findings indicate an important role of exogenous mitochondrial trafficking from BM stromal cells to AML cells as well as endogenous mitochondrial fission and mitophagy in the compensatory adaptation of leukemia cells to energetic stress in the BM microenvironment.
  4. Clin Adv Hematol Oncol. 2021 Sep;19(9): 556-558
  5. FEBS J. 2021 Sep 05.
      Homeostasis in the blood system is maintained by the balance between self-renewing stem cells and non-stem cells. To promote self-renewal, transcriptional regulators maintain epigenetic information during multiple rounds of cell division. Mutations in such transcriptional regulators cause aberrant self-renewal, leading to leukemia. MOZ, a histone acetyltransferase, and MLL, a histone methyltransferase, are transcriptional regulators that promote the self-renewal of hematopoietic stem cells. Gene rearrangements of MOZ and MLL generate chimeric genes encoding fusion proteins that function as constitutively active forms. These MOZ- and MLL-fusion proteins constitutively activate transcription of their target genes and cause aberrant self-renewal in committed hematopoietic progenitors, which normally do not self-renew. Recent progress in the field suggests that MOZ and MLL are part of a transcriptional activation system that activates the transcription of genes with non-methylated CpG-rich promoters. The non-methylated state of CpGs is normally maintained during cell divisions from the mother cell to the daughter cells. Thus, the MOZ/MLL-mediated transcriptional activation system replicates the expression profile of mother cells in daughter cells by activating the transcription of genes previously transcribed in the mother cell. This review summarizes the functions of the components of the MOZ/MLL-mediated transcriptional activation system and their roles in the promotion of self-renewal.
    Keywords:  MLL; MORF; MOZ; histone acetyltransferase; histone methyltransferase; leukemia; protein complex; self-renewal; transcription; unmethylated CpG
  6. Leukemia. 2021 Sep 08.
      The evaluation of bone marrow morphology by experienced hematopathologists is essential in the diagnosis of acute myeloid leukemia (AML); however, it suffers from a lack of standardization and inter-observer variability. Deep learning (DL) can process medical image data and provides data-driven class predictions. Here, we apply a multi-step DL approach to automatically segment cells from bone marrow images, distinguish between AML samples and healthy controls with an area under the receiver operating characteristic (AUROC) of 0.9699, and predict the mutation status of Nucleophosmin 1 (NPM1)-one of the most common mutations in AML-with an AUROC of 0.92 using only image data from bone marrow smears. Utilizing occlusion sensitivity maps, we observed so far unreported morphologic cell features such as a pattern of condensed chromatin and perinuclear lightening zones in myeloblasts of NPM1-mutated AML and prominent nucleoli in wild-type NPM1 AML enabling the DL model to provide accurate class predictions.
  7. Curr Hematol Malig Rep. 2021 Sep 09.
      PURPOSE OF REVIEW: Despite recent advances in the treatment of de novo acute myeloid leukemia (AML), AML arising from antecedent chronic myelomonocytic leukemia (CMML) continues to have dismal outcomes. While the unique biological drivers of CMML and subsequent leukemic transformation (LT) have been revealed with advances in molecular characterization, this has not yet translated to the bedside. Here, we review these biologic drivers, outcomes with current therapies, and rationale avenues of future investigation specifically in blast phase CMML (CMML-BP).RECENT FINDINGS: CMML-BP outcomes are studied as an aggregate with more common categories of AML with myelodysplasia-related changes (AML-MRCs) or the even broader category of secondary AML (sAML), which illustrates the crux of the problem. While a modest survival advantage with allogeneic hematopoietic stem cell transplant exists, the difficulty is bridging patients to transplant and managing patients that require an allograft-sparing approach. Limited data suggest that short-lived remissions can be obtained employing CPX-351 or venetoclax-based lower intensity combination therapy. Promising future strategies include repurposing cladribine, exploiting the supportive role of dendritic cell subsets with anti-CD123 therapies, MCL-1 inhibition, dual MEK/PLK1 inhibition, FLT3 inhibition in RAS-mutated and CBL-mutated subsets, and immune therapies targeting novel immune checkpoint molecules such as the leukocyte immunoglobulin-like receptor B4 (LILRB4), an immune-modulatory transmembrane protein restrictively expressed on monocytic cells. The successful management of an entity as unique as CMML-BP will require a cooperative, concerted effort to design and conduct clinical trials dedicated to this rare form of sAML.
    Keywords:  Allogeneic stem cell transplant; Blast phase; CPX-351; Chronic myelomonocytic leukemia; Secondary acute myeloid leukemia; Venetoclax
  8. Oncotarget. 2021 Aug 31. 12(18): 1763-1779
      Acute myeloid leukemia (AML) with fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) relapses with new chromosome abnormalities following chemotherapy, implicating genomic instability. Error-prone alternative non-homologous end-joining (Alt-NHEJ) DNA double-strand break (DSB) repair is upregulated in FLT3-ITD-expresssing cells, driven by c-Myc. The serine/threonine kinase Pim-1 is upregulated downstream of FLT3-ITD, and inhibiting Pim increases topoisomerase 2 (TOP2) inhibitor chemotherapy drug induction of DNA DSBs and apoptosis. We hypothesized that Pim inhibition increases DNA DSBs by downregulating Alt-NHEJ, also decreasing genomic instability. Alt-NHEJ activity, measured with a green fluorescent reporter construct, increased in FLT3-ITD-transfected Ba/F3-ITD cells treated with TOP2 inhibitors, and this increase was abrogated by Pim kinase inhibitor AZD1208 co-treatment. TOP2 inhibitor and AZD1208 co-treatment downregulated cellular and nuclear expression of c-Myc and Alt-NHEJ repair pathway proteins DNA polymerase θ, DNA ligase 3 and XRCC1 in FLT3-ITD cell lines and AML patient blasts. ALT-NHEJ protein downregulation was preceded by c-Myc downregulation, inhibited by c-Myc overexpression and induced by c-Myc knockdown or inhibition. TOP2 inhibitor treatment increased chromosome breaks in metaphase spreads in FLT3-ITD-expressing cells, and AZD1208 co-treatment abrogated these increases. Thus Pim kinase inhibitor co-treatment both enhances TOP2 inhibitor cytotoxicity and decreases TOP2 inhibitor-induced genomic instability in cells with FLT3-ITD.
    Keywords:  FLT3 internal tandem duplication; Pim kinase; alternative non-homologous end-joining DNA repair; genomic instability; topoisomerase 2 inhibitors
  9. Leuk Lymphoma. 2021 Sep 09. 1-13
      TP53-aberrant myelodysplastic syndrome and acute myeloid leukemia have dismal outcomes. Here, we define the clinico-genomic landscape of TP53 disruptions in 40 patients and employ clonal dynamic modeling to map the mutational hierarchy against clinical outcomes. Most TP53 mutations (45.2%) localized to the L3 loop or LSH motif of the DNA-binding domain. TP53 disruptions had high co-occurrence with mutations in epigenetic regulators, spliceosome machinery, and cohesin complex and low co-occurrence with mutations in proliferative signaling genes. Ancestral and descendant TP53 mutations constituted measurable residual disease and fueled relapse. High mutant TP53 gene dosage predicted low durability of remission. The median overall survival (OS) was 280 days. Hypomethylating agent-based therapy served as an effective bridge to transplant, leading to improved median OS compared to patients who did not receive a transplant (14.7 vs. 5.1 months). OS was independent of the genomic location of TP53 disruption, which has implications for rational therapeutic design.
    Keywords:  Myelodysplastic syndrome; TP53; acute myeloid leukemia; clonal dynamics; hematopoietic stem cell; hypomethylating agents
  10. Blood Adv. 2021 Sep 10. pii: bloodadvances.2021004856. [Epub ahead of print]
      Background Although somatic mutations influence the pathogenesis, phenotype, and outcome of myeloproliferative neoplasms (MPN), little is known about their impact on molecular response to cytoreductive treatment. Methods We performed targeted next-generation sequencing (NGS) on 202 pre-treatment samples obtained from patients with MPN enrolled in the DALIAH trial (randomized controlled phase III clinical trial, NCT01387763) and 135 samples obtained after 24 months of therapy with recombinant interferon-alpha (IFNα) or hydroxyurea (HU). The primary aim was to evaluate the association between complete clinicohematologic response (CHR) at 24 months and molecular response through sequential assessment of 120 genes using NGS. Results Among JAK2-mutated patients treated with IFNα, those with CHR had a greater reduction in the JAK2 variant allele frequency (VAF) (median 0.29 to 0.07; p<0.0001) compared with those not achieving CHR (median 0.27 to 0.14; p<0.0001). In contrast, the CALR VAF did not significantly decline in neither those achieving CHR nor those not achieving CHR. Treatment-emergent mutations in DNMT3A were observed more commonly in patients treated with IFNα compared with HU, p=0.04. Furthermore, treatment-emergent DNMT3A-mutations were significantly enriched in IFNα treated patients not attaining CHR, p=0.02. A mutation in TET2, DNMT3A, or ASXL1 was significantly associated with prior stroke (age-adjusted OR=5.29 [95% CI, 1.59-17.54]; p=0.007) as was a mutation in TET2 alone (age-adjusted OR=3.03 [95% CI, 1.03-9.01]; p=0.044). Conclusion At 24 months, we found mutation-specific response patterns to IFNα: (1) JAK2- and CALR-mutated MPN demonstrated distinct molecular responses and (2) DNMT3A-mutated clones/subclones emerged on treatment.
  11. Blood. 2021 Sep 05. pii: blood.2020005693. [Epub ahead of print]
      Post-transcriptional regulation has emerged as a driver for leukemia development and an avenue for therapeutic targeting. Among post-transcriptional processes, alternative polyadenylation (APA) is globally dysregulated across cancer types. However, limited studies have focused on the prevalence and role of APA in myeloid leukemia. Furthermore, it is poorly understood how altered poly(A) site (PAS) usage of individual genes contributes to malignancy or whether targeting global APA patterns might alter oncogenic potential. In this study, we examined global APA dysregulation in acute myeloid leukemia (AML) patients by performing 3' Region Extraction And Deep Sequencing (3'READS) on a subset of AML patient samples along with healthy hematopoietic stem and progenitor cells (HSPCs) and by analyzing publicly available data from a broad AML patient cohort. We show that patient cells exhibit global 3' untranslated region (UTR) shortening and coding sequence (CDS) lengthening due to differences in PAS usage. Among APA regulators, expression of FIP1L1, one of the core cleavage and polyadenylation factors, correlated with the degree of APA dysregulation in our 3'READS dataset. Targeting global APA by FIP1L1 knockdown reversed the global trends seen in patients. Importantly, FIP1L1 knockdown induced differentiation of t(8;21) cells by promoting 3'UTR lengthening and downregulation of the fusion oncoprotein AML1-ETO. In non-t(8;21) cells, FIP1L1 knockdown also promoted differentiation by attenuating mTORC1 signaling and reducing MYC protein levels. Our study provides mechanistic insights into the role of APA in AML pathogenesis and indicates that targeting global APA patterns can overcome the differentiation block of AML patients.
  12. Cell Metab. 2021 Sep 07. pii: S1550-4131(21)00373-9. [Epub ahead of print]33(9): 1719-1720
      Supporting the notion that cell lineage is a key determinant of cancer cell metabolism, Jun et al. (2021) identify a selective requirement for pyruvate dehydrogenase (PDH) activity in T cells and T cell leukemia, but not hematopoietic stem cells (HSCs) or myeloid leukemia, in this issue of Cell Metabolism.
  13. Blood Adv. 2021 Sep 08. pii: bloodadvances.2021004726. [Epub ahead of print]
      During aging, hematopoietic stem cell (HSC) function wanes with important biological and clinical implications for benign and malignant hematology, and other co-morbidities, such as cardiovascular disease. However, the molecular mechanisms regulating HSC aging remain incompletely defined. GATA2 haploinsufficiency driven clinical syndromes initially result in primary immunodeficiencies and routinely evolve into hematologic malignancies on acquisition of further epigenetic mutations in both young and older patients. Using a conditional mouse model of Gata2 haploinsufficiency, we discover that during aging Gata2 promotes HSC proliferation, monocytosis, and loss of the common lymphoid progenitor. Aging of Gata2 haploinsufficient mice also offsets enhanced HSC apoptosis and decreased granulocyte-macrophage progenitor number normally observed in young Gata2 haploinsufficient mice. Transplantation of elderly Gata2 haploinsufficient HSCs impairs HSC function with evidence of myeloid bias. Our data demonstrate that Gata2 regulates HSC aging and suggest the mechanisms by which Gata2 mediated HSC aging has an impact on the evolution of malignancies in GATA2 haploinsufficiency syndromes.
  14. Sci Rep. 2021 Sep 09. 11(1): 18012
      DNAM-1 is reportedly expressed on cytotoxic T and NK cells and, upon interaction with its ligands CD112 and CD155, plays an important role in tumor immunosurveillance. It has also been reported to be functionally expressed by myeloid cells, but expression and function on malignant cells of the myeloid lineage have not been studied so far. Here we analyzed expression of DNAM-1 in leukemic cells of acute myeloid leukemia (AML) patients. We found substantial levels of DNAM-1 to be expressed on leukemic blasts in 48 of 62 (> 75%) patients. Interaction of DNAM-1 with its ligands CD112 and CD155 induced release of the immunomodulatory cytokines IL-6, IL-8 IL-10 and TNF-α by AML cells and DNAM-1 expression correlated with a more differentiated phenotype. Multivariate analysis did not show any association of DNAM-1 positivity with established risk factors, but expression was significantly associated with clinical disease course: patients with high DNAM-1 surface levels had significantly longer progression-free and overall survival compared to DNAM-1low patients, independently whether patients had undergone allogenic stem cell transplantation or not. Together, our findings unravel a functional role of DNAM-1 in AML pathophysiology and identify DNAM-1 as a potential novel prognostic maker in AML.
  15. Molecules. 2021 Aug 31. pii: 5300. [Epub ahead of print]26(17):
      Histone methyltransferase DOT1L catalyzes mono-, di- and trimethylation of histone 3 at lysine residue 79 (H3K79) and hypermethylation of H3K79 has been linked to the development of acute leukemias characterized by the MLL (mixed-lineage leukemia) rearrangements (MLLr cells). The inhibition of H3K79 methylation inhibits MLLr cells proliferation, and an inhibitor specific for DOT1L, pinometostat, was in clinical trials (Phase Ib/II). However, the compound showed poor pharmacological properties. Thus, there is a need to find new potent inhibitors of DOT1L for the treatment of rearranged leukemias. Here we present the design, synthesis, and biological evaluation of a small molecule that inhibits in the nM level the enzymatic activity of hDOT1L, H3K79 methylation in MLLr cells with comparable potency to pinometostat, associated with improved metabolic stability and a characteristic cytostatic effect.
    Keywords:  DOT1L; HMT inhibitors; MLL rearranged leukemia; bisubstrates; histone methylation; rational drug design
  16. J Natl Cancer Inst. 2021 Sep 07. pii: djab184. [Epub ahead of print]
      Treatment-free remission (TFR) is a goal for patients with chronic myeloid leukemia (CML). Functional outcomes after discontinuing Tyrosine Kinase Inhibitor (TKI) treatment have not been described. PROMIS patient-reported outcome measures (PROMs) of social, physical, cognitive, and sexual function were assessed over 36 months in 172 adult patients with chronic phase CML from 14 sites at baseline (on TKI) and after discontinuation. Linear mixed-effects models described the average trajectories for each PROM after discontinuation and in those who restarted TKI. Of 112 patients in TFR at 12 months, 103 (92.0%) had a ≥ 3-point improvement in social function, 80 (71.4%) in social isolation, 11 (9.8%) in satisfaction with sex life, 4 (3.6%) in physical function, and no patients had a ≥ 3-point improvement in cognitive function or interest in sexual activity. Patients' scores worsened after restarting TKI. This novel information on functional outcomes in TFR can help guide patient and clinician decision-making.
  17. Cancers (Basel). 2021 Aug 30. pii: 4372. [Epub ahead of print]13(17):
      This study was directed to characterize the role of glutamine in the modulation of the response of chronic myeloid leukemia (CML) cells to low oxygen, a main condition of hematopoietic stem cell niches of bone marrow. Cells were incubated in atmosphere at 0.2% oxygen in the absence or the presence of glutamine. The absence of glutamine markedly delayed glucose consumption, which had previously been shown to drive the suppression of BCR/Abl oncoprotein (but not of the fusion oncogene BCR/abl) in low oxygen. Glutamine availability thus emerged as a key regulator of the balance between the pools of BCR/Abl protein-expressing and -negative CML cells endowed with stem/progenitor cell potential and capable to stand extremely low oxygen. These findings were confirmed by the effects of the inhibitors of glucose or glutamine metabolism. The BCR/Abl-negative cell phenotype is the best candidate to sustain the treatment-resistant minimal residual disease (MRD) of CML because these cells are devoid of the molecular target of the BCR/Abl-active tyrosine kinase inhibitors (TKi) used for CML therapy. Therefore, the treatments capable of interfering with glutamine action may result in the reduction in the BCR/Abl-negative cell subset sustaining MRD and in the concomitant rescue of the TKi sensitivity of CML stem cell potential. The data obtained with glutaminase inhibitors seem to confirm this perspective.
    Keywords:  chronic myeloid leukemia; glucose; glutamine; low oxygen; minimal residual disease; stem cell niche
  18. Blood Adv. 2021 Sep 14. 5(17): 3492-3496
      The basis for acquired resistance to JAK inhibition in patients with JAK2-driven hematologic malignancies is not well understood. We report a patient with a myeloproliferative neoplasm (MPN) with a BCR activator of RhoGEF and GTPase (BCR)-JAK2 fusion with initial hematologic response to ruxolitinib who rapidly developed B-lymphoid blast transformation. We analyzed pre-ruxolitinib and blast transformation samples using genome sequencing, DNA mate-pair sequencing (MPseq), RNA sequencing (RNA-seq), and chromosomal microarray to characterize possible mechanisms of resistance. No resistance mutations in the BCR-JAK2 fusion gene or transcript were identified, and fusion transcript expression levels remained stable. However, at the time of blast transformation, MPseq detected a new IKZF1 copy-number loss, which is predicted to result in loss of normal IKZF1 protein translation. RNA-seq revealed significant upregulation of genes negatively regulated by IKZF1, including IL7R and CRLF2. Disease progression was also characterized by adaptation to an activated B-cell receptor (BCR)-like signaling phenotype, with marked upregulation of genes such as CD79A, CD79B, IGLL1, VPREB1, BLNK, ZAP70, RAG1, and RAG2. In summary, IKZF1 deletion and a switch from cytokine dependence to activated BCR-like signaling phenotype represent putative mechanisms of ruxolitinib resistance in this case, recapitulating preclinical data on resistance to JAK inhibition in CRLF2-rearranged Philadelphia chromosome-like acute lymphoblastic leukemia.
  19. Leukemia. 2021 Sep 08.
      Insufficiency of polycomb repressive complex 2 (PRC2), which trimethylates histone H3 at lysine 27, is frequently found in primary myelofibrosis and promotes the development of JAK2V617F-induced myelofibrosis in mice by enhancing the production of dysplastic megakaryocytes. Polycomb group ring finger protein 1 (Pcgf1) is a component of PRC1.1, a non-canonical PRC1 that monoubiquitylates H2A at lysine 119 (H2AK119ub1). We herein investigated the impact of PRC1.1 insufficiency on myelofibrosis. The deletion of Pcgf1 in JAK2V617F mice strongly promoted the development of lethal myelofibrosis accompanied by a block in erythroid differentiation. Transcriptome and chromatin immunoprecipitation sequence analyses showed the de-repression of PRC1.1 target genes in Pcgf1-deficient JAK2V617F hematopoietic progenitors and revealed Hoxa cluster genes as direct targets. The deletion of Pcgf1 in JAK2V617F hematopoietic stem and progenitor cells (HSPCs), as well as the overexpression of Hoxa9, restored the attenuated proliferation of JAK2V617F progenitors. The overexpression of Hoxa9 also enhanced JAK2V617F-mediated myelofibrosis. The expression of PRC2 target genes identified in PRC2-insufficient JAK2V617F HSPCs was not largely altered in Pcgf1-deleted JAK2V617F HSPCs. The present results revealed a tumor suppressor function for PRC1.1 in myelofibrosis and suggest that PRC1.1 insufficiency has a different impact from that of PRC2 insufficiency on the pathogenesis of myelofibrosis.
  20. Blood Adv. 2021 Sep 07. pii: bloodadvances.2020003969. [Epub ahead of print]
      RUNX1 is essential for the generation of hematopoietic stem cells (HSCs). Runx1 null mouse embryos lack definitive hematopoiesis and die in mid-gestation. However, even though zebrafish embryos with a runx1 W84X mutation have defects in early definitive hematopoiesis, some runx1W84X/W84X embryos can develop to fertile adults with blood cells of multi-lineages, raising the possibility that HSCs can emerge without RUNX1. Here, using three new zebrafish runx1-/- lines we uncovered the compensatory mechanism for runx1-independent hematopoiesis. We show that, in the absence of a functional runx1, a cd41-GFP+ population of hematopoietic precursors still emerge from the hemogenic endothelium and can colonize the hematopoietic tissues of the mutant embryos. Single-cell RNA sequencing of the cd41-GFP+ cells identified a set of runx1-/--specific signature genes during hematopoiesis. Significantly, gata2b, which normally acts upstream of runx1 for the generation of HSCs, was increased in the cd41-GFP+ cells in runx1- /- embryos. Interestingly, genetic inactivation of both gata2b and its paralog, gata2a, did not affect hematopoiesis. However, knocking out runx1 and any three of the four alleles of gata2a and gata2b abolished definitive hematopoiesis. Gata2 expression was also upregulated in hematopoietic cells in Runx1-/- mice, suggesting the compensatory mechanism is conserved. Our findings indicate that RUNX1 and GATA2 serve redundant roles for HSC production, acting as each other's safeguard.
  21. Leuk Lymphoma. 2021 Sep 08. 1-10
      Implications of creatine kinase (CK) elevation, a frequent complication of tyrosine kinase inhibitor (TKI) treatment for chronic myeloid leukemia (CML), on its key treatment outcomes (overall survival (OS) and event-free survival (EFS)), remain unknown. In this single center, retrospective study on 283 chronic phase CML patients on first-line TKI (median follow-up of 8.8 years), 71.7% patients had hyperCKemia with no difference in incidence between imatinib and second generation TKIs (SG-TKIs). In multivariable Cox regression analysis, hyperCKemia was associated with better OS and intermediate- and high-Sokal risk score with worse OS. In multivariable Cox regression for EFS, hyperCKemia and treatment with SG-TKI were associated with improved EFS while intermediate or high Sokal index and higher comorbidities showed worse EFS. Our study provides an evidence on the prognostic value of hyperCKemia in CML and informs clinicians not to change TKI based solely on laboratory elevations of CK.
    Keywords:  Chronic myeloid leukemia; creatine kinase; tyrosine kinase inhibitors
  22. Sci Rep. 2021 Sep 06. 11(1): 17702
      A subset of essential thrombocythemia (ET) cases are negative for disease-defining mutations on JAK2, MPL, and CALR and defined as triple negative (TN). The lack of recurrent mutations in TN-ET patients makes its pathogenesis ambiguous. Here, we screened 483 patients with suspected ET in a single institution, centrally reviewed bone marrow specimens, and identified 23 TN-ET patients. Analysis of clinical records revealed that TN-ET patients were mostly young female, without a history of thrombosis or progression to secondary myelofibrosis and leukemia. Sequencing analysis and human androgen receptor assays revealed that the majority of TN-ET patients exhibited polyclonal hematopoiesis, suggesting a possibility of reactive thrombocytosis in TN-ET. However, the serum levels of thrombopoietin (TPO) and interleukin-6 in TN-ET patients were not significantly different from those in ET patients with canonical mutations and healthy individuals. Rather, CD34-positive cells from TN-ET patients showed a capacity to form megakaryocytic colonies, even in the absence of TPO. No signs of thrombocytosis were observed before TN-ET development, denying the possibility of hereditary thrombocytosis in TN-ET. Overall, these findings indicate that TN-ET is a distinctive disease entity associated with polyclonal hematopoiesis and is paradoxically caused by hematopoietic stem cells harboring a capacity for cell-autonomous megakaryopoiesis.
  23. Oncogene. 2021 Sep 06.
      Leukemia patients bearing the t(4;11)(q21;q23) translocations can be divided into two subgroups: those expressing both reciprocal fusion genes, and those that have only the MLL-AF4 fusion gene. Moreover, a recent study has demonstrated that patients expressing both fusion genes have a better outcome than patients that are expressing the MLL-AF4 fusion protein alone. All this may point to a clonal process where the reciprocal fusion gene AF4-MLL could be lost during disease progression, as this loss may select for a more aggressive type of leukemia. Therefore, we were interested in unraveling the decisive role of the AF4-MLL fusion protein at an early timepoint of disease development. We designed an experimental model system where the MLL-AF4 fusion protein was constitutively expressed, while an inducible AF4-MLL fusion gene was induced for only 48 h. Subsequently, we investigated genome-wide changes by RNA- and ATAC-Seq experiments at distinct timepoints. These analyses revealed that the expression of AF4-MLL for only 48 h was sufficient to significantly change the genomic landscape (transcription and chromatin) even on a longer time scale. Thus, we have to conclude that the AF4-MLL fusion protein works through a hit-and-run mechanism, probably necessary to set up pre-leukemic conditions, but being dispensable for later disease progression.
  24. Leuk Res. 2021 Aug 31. pii: S0145-2126(21)00202-2. [Epub ahead of print]110 106701
      AML with myelodysplasia-related changes (AML-MRC) is a subtype of AML known to have adverse prognosis. The karyotype abnormalities in AML-MRC have been well established; however, relatively little has been known about the role of gene mutation profiles by next generation sequencing. 177 AML patients (72 AML-MRC and 105 non-MRC AML) were analyzed by NGS panel covering 53 AML related genes. AML-MRC showed statistically significantly higher frequency of TP53 mutation, but lower frequencies of mutations in NPM1, FLT3-ITDLow, FLT3-ITDHigh, FLT3-TKD, NRAS, and PTPN11 than non-MRC AML. Supervised tree-based classification models including Decision tree, Random forest, and XGboost, and logistic regression were used to evaluate if the mutation profiles could be used to aid the diagnosis of AML-MRC. All methods showed good accuracy in differentiating AML-MRC from non-MRC AML with AUC (area under curve) of ROC ranging from 0.69 to 0.78. Additionally, logistic regression indicated 3 independent factors (age and mutations of TP53 and FLT3) could aid the diagnosis AML-MRC. Using weighted factors, a AML-MRC risk scoring equation was established for potential application in clinical setting: +1x(Age ≥ 65) + 3 x (TP53 mutation) - 2 x (FLT3 mutation). Using a cutoff score of 0, the accuracy of the risk score was 0.76 with sensitivity of 0.77 and specificity of 0.75 for predicting the diagnosis of AML-MRC. Further studies with larger sample sizes are warranted to further evaluate the potential of using gene mutation profiles to aid the diagnosis of AML-MRC.
    Keywords:  AML with MRC; FLT3; Machine learning; NGS; Supervised classification; TP53
  25. Nat Commun. 2021 Sep 09. 12(1): 5248
      The HRAS, NRAS, and KRAS genes are collectively mutated in a fifth of all human cancers. These mutations render RAS GTP-bound and active, constitutively binding effector proteins to promote signaling conducive to tumorigenic growth. To further elucidate how RAS oncoproteins signal, we mined RAS interactomes for potential vulnerabilities. Here we identify EFR3A, an adapter protein for the phosphatidylinositol kinase PI4KA, to preferentially bind oncogenic KRAS. Disrupting EFR3A or PI4KA reduces phosphatidylinositol-4-phosphate, phosphatidylserine, and KRAS levels at the plasma membrane, as well as oncogenic signaling and tumorigenesis, phenotypes rescued by tethering PI4KA to the plasma membrane. Finally, we show that a selective PI4KA inhibitor augments the antineoplastic activity of the KRASG12C inhibitor sotorasib, suggesting a clinical path to exploit this pathway. In sum, we have discovered a distinct KRAS signaling axis with actionable therapeutic potential for the treatment of KRAS-mutant cancers.
  26. Br J Haematol. 2021 Sep 07.
      Patients with acute myeloid leukaemia (AML) have a five-year survival rate of 28·7%. Natural killer (NK)-cell have anti-leukaemic activity. Here, we report on a series of 13 patients with high-risk R/R AML, treated with repeated infusions of double-bright (CD56bright /CD16bright ) expanded NK cells at an academic centre in Brazil. NK cells from HLA-haploidentical donors were expanded using K562 feeder cells, modified to express membrane-bound interleukin-21. Patients received FLAG, after which cryopreserved NK cells were thawed and infused thrice weekly for six infusions in three dose cohorts (106 -107 cells/kg/infusion). Primary objectives were safety and feasibility. Secondary endpoints included overall response (OR) and complete response (CR) rates at 28-30 days after the first infusion. Patients received a median of five prior lines of therapy, seven with intermediate or adverse cytogenetics, three with concurrent central nervous system (CNS) leukaemia, and one with concurrent CNS mycetoma. No dose-limiting toxicities, infusion-related fever, or cytokine release syndrome were observed. An OR of 78·6% and CR of 50·0% were observed, including responses in three patients with CNS disease and clearance of a CNS mycetoma. Multiple infusions of expanded, cryopreserved NK cells were safely administered after intensive chemotherapy in high-risk patients with R/R AML and demonstrated encouraging outcomes.
    Keywords:  CNS leukaemia; NK cell; R/R AML; adoptive immunotherapy
  27. Blood. 2021 Sep 05. pii: blood.2021011354. [Epub ahead of print]
      Inherited predisposition to myeloid malignancies is more common than previously appreciated. We analyzed the whole-exome sequencing data of paired leukemia and skin biopsy samples from 391 adult patients from the Beat AML 1.0 consortium. Using the 2015 ACMG guidelines for variant interpretation, we curated 1,547 unique variants from 228 genes. The pathogenic/likely pathogenic (P/LP) germline variants were identified in 53 AML patients (13.6%) in 34 genes. 41% of variants were in DNA damage response genes, and the most frequently mutated genes were CHEK2 (8 patients) and DDX41 (7 patients). 44% of the pathogenic germline variants were in genes considered clinically actionable (tier 1). Pathogenic germline variants were also found in new candidate genes (DNAH5, DNAH9, DNMT3A, SUZ12). No strong correlation was found between the germline mutational rate and age of AML onset. Among 49 patients who have a reported history of at least one family member affected with hematological malignancies, six patients harbored known P/LP germline variants and the remaining patients had at least one variant of uncertain significance, suggesting a need for further functional validation studies. Using CHEK2 as an example, we show that three-dimensional protein modeling can be one of the effective methodologies to prioritize variants of unknown significance for functional studies. Further, we evaluated an in-silico approach that applies ACMG/AMP curation in an automated manner using the tool for assessment and prioritization in exome studies (TAPES), which can minimize manual curation time for variants. Overall, our findings suggest a need to comprehensively understand the predisposition potential of many germline variants in order to enable closer monitoring for disease management and treatment interventions for affected patients and families.
  28. Elife. 2021 Sep 06. pii: e66198. [Epub ahead of print]10
      In cancer, telomere maintenance is critical for the development of replicative immortality. Using genome sequences from the Cancer Cell Line Encyclopedia and Genomics of Drug Sensitivity in Cancer Project, we calculated telomere content across 1,299 cancer cell lines. We find that telomerase reverse transcriptase (TERT) expression correlates with telomere content in lung, central nervous system, and leukemia cell lines. Using CRISPR/Cas9 screening data, we show that lower telomeric content is associated with dependency of CST telomere maintenance genes. Increased dependencies of shelterin members are associated with wild-type TP53 status. Investigating the epigenetic regulation of TERT, we find widespread allele-specific expression in promoter-wildtype contexts. TERT promoter-mutant cell lines exhibit hypomethylation at PRC2-repressed regions, suggesting a cooperative global epigenetic state in the reactivation of telomerase. By incorporating telomere content with genomic features across comprehensively characterized cell lines, we provide further insights into the role of telomere regulation in cancer immortality.
    Keywords:  cancer biology; computational biology; human; systems biology
  29. Blood. 2021 Sep 09. pii: blood.2021010745. [Epub ahead of print]
      Hematopoietic stem cells (HSCs) sustain blood cell homeostasis throughout life and can regenerate all blood lineages following transplantation. Despite this clear functional definition, highly enriched isolation of human HSCs can currently only be achieved through combinatorial assessment of multiple surface antigens. While several transgenic HSC reporter mouse strains have been described, no analogous approach to prospectively isolate human HSCs has been reported. To identify genes with the most selective expression in human HSCs, we profiled population- and single-cell transcriptomes of un-expanded and ex vivo cultured cord blood-derived HSPCs as well as peripheral blood, adult bone marrow, and fetal liver. Based on these analyses, we propose the master transcription factor HLF (Hepatic Leukemia Factor) as one of the most specific HSC marker genes. To directly track its expression in human hematopoietic cells, we developed a genomic HLF reporter strategy, capable of selectively labeling the most immature blood cells based on a single engineered parameter. Most importantly, HLF-expressing cells comprise all of the stem cell activity in culture and in vivo during serial transplantation. Taken together, these results experimentally establish HLF as a defining gene of the human hematopoietic stem cell state and outline a new approach to continuously mark these cells with high fidelity.
  30. Blood Adv. 2021 Sep 07. pii: bloodadvances.2021004750. [Epub ahead of print]
      As part of the inflammatory response by macrophages, Irg1 is induced resulting in millimolar quantities of itaconate being produced. This immunometabolite remodels the macrophage metabolome and acts as an antimicrobial agent when excreted. Itaconate is not synthesized within the erythron, but instead may be acquired from central macrophages within the erythroid island. Previously we reported that itaconate inhibits hemoglobinzation of developing erythroid cells. Herein we demonstrate that this is accomplished by inhibition of tetrapyrrole synthesis. In differentiating erythroid precursors, cellular heme and protoporphyrin IX synthesis are reduced by itaconate at an early step in the pathway. In addition, itaconate causes global alterations in cellular metabolite pools resulting in elevated levels of succinate, 2-hydroxyglutarate, pyruvate, glyoxylate, and intermediates of glycolytic shunts. Itaconate taken up by the developing erythron can be converted to itaconyl-CoA by the enzyme succinyl-CoA:glutarate-CoA transferase. Propionyl-CoA, propionyl-carnitine, methylmalonic acid, heptadecanoic acid and nonanoic acid, as well as the aliphatic amino acids threonine, valine, methionine, and isoleucine are increased, likely due to the impact of endogenous itaconyl-CoA synthesis. We further show that itaconyl-CoA is a competitive inhibitor of the erythroid-specific 5-aminolevulinate synthase (ALAS2), the first and rate-limiting step in heme synthesis. These findings strongly support our hypothesis that the inhibition of heme synthesis observed in chronic inflammation is mediated not only by iron limitation, but also by limitation of tetrapyrrole synthesis at the point of ALAS2 catalysis by itaconate. Thus, we propose that macrophage-derived itaconate promotes anemia during an inflammatory response in the erythroid compartment.
  31. Br J Haematol. 2021 Sep 08.
      The doubling time (DT) of the BCR-ABL1 quantitative polymerase chain reaction (qPCR) transcript level reflects the re-growing fraction of leukaemic cells after discontinuation of tyrosine kinase inhibitor (TKI). The present study analyzed monthly DT within six months after imatinib discontinuation in 131 patients. Monthly DT was calculated as x = ln(2)/K, where x is the DT and K is the fold BCR-ABL1 change from the previous value divided by the number of days between each measurement. The optimal DT value was determined as 12·75 days at two months using a recursive partitioning method. The patients were stratified into three groups: the high-risk group (DT<12·75 days but >0, with rapidly proliferating chronic myeloid leukaemia (CML) cells; n = 26) showed the lowest molecular relapse-free survival (mRFS) of 7·7% at 12 months, compared to 53·6% in the intermediate-risk group (DT≥12·75 days, with slowly proliferating CML cells; n = 16) or 90·0% in the low-risk group (DT≤0, i.e., without proliferating CML cells; n = 71; P < 0·001). Monthly assessment of DT helps identify high-risk patients for treatment-free remission failure with an imminent risk of molecular recurrence, and to define low-risk patients who can be spared the frequent monitoring of monthly molecular tests.
    Keywords:  CML; IMATINIB; TKI discontinuation; doubling time; treatment-free remission
  32. Blood Adv. 2021 Sep 10. pii: bloodadvances.2021004319. [Epub ahead of print]
      Individual comorbidities have distinct contributions to non-relapse mortality (NRM) following allogeneic hematopoietic cell transplantation (allo-HCT). We studied the impact of comorbidities both individually and in combination in a single-center cohort of 573 adult patients who underwent CD34-selected allo-HCT following myeloablative conditioning. Pulmonary disease, moderate to severe hepatic comorbidity, cardiac disease of any type, and renal dysfunction were associated with increased NRM in multivariable Cox regression models. A Simplified Comorbidity Index (SCI) composed of the four comorbidities predictive of NRM, as well as age > 60 years, stratified patients into five groups with a stepwise increase in NRM. NRM rates ranged from 11.4% to 49.9% by stratum, with adjusted hazard ratios of 1.84, 2.59, 3.57, and 5.38. The SCI was also applicable in an external cohort of 230 patients who underwent allo-HCT with unmanipulated grafts following intermediate-intensity conditioning. The area under the ROC curve (AUC) of the SCI for 1-year NRM was 70.3 and 72.0 over the development and external-validation cohorts, respectively; corresponding AUCs of the Hematopoietic Cell Transplantation-specific Comorbidity Index (HCT-CI) were 61.7 and 65.7. In summary, a small set of comorbidities, aggregated into the Simplified Comorbidity Index, are highly predictive of NRM. The new index stratifies patients into distinct risk groups, was validated in an external cohort, and provides higher discrimination than the HCT-CI.
  33. Blood Adv. 2021 09 14. 5(17): 3407-3417
      Recent compelling evidence showed that innate immune effector cells could recognize allogeneic grafts and prime an adaptive immune response. Signal regulatory protein α (SIRPα) is an immunoglobulin superfamily receptor that is expressed on myeloid cells; the interaction between SIRPα and its ubiquitously expressed ligand CD47 elicits an inhibitory signal that suppresses macrophage phagocytic function. Additional studies showed that donor-recipient mismatch in SIRPα variants might activate monocytic allorecognition, possibly as the result of non-self SIRPα-CD47 interaction. However, the frequency of SIRPα variation and its role in hematopoietic stem cell transplantation (HSCT) remains unexplored. We studied 350 patients with acute myeloid leukemia/myelodysplastic syndrome who underwent HLA-matched related HSCT and found that SIRPα allelic mismatches were present in 39% of transplantation pairs. SIRPα variant mismatch was associated with a significantly higher rate of chronic graft-versus-host disease (GVHD; hazard ratio [HR], 1.5; P = .03), especially de novo chronic GVHD (HR, 2.0; P = .01), after adjusting for other predictors. Those with mismatched SIRPα had a lower relapse rate (HR, 0.6; P = .05) and significantly longer relapse-free survival (RFS; HR, 0.6; P = .04). Notably, the effect of SIRPα variant mismatch on relapse protection was most pronounced early after HSCT and in patients who were not in remission at HSCT (cumulative incidence, 73% vs 54%; HR, 0.5; P = .01). These findings show that SIRPα variant mismatch is associated with HSCT outcomes, possibly owing to innate allorecognition. SIRPα variant matching could provide valuable information for donor selection and risk stratification in HSCT.