bims-tremyl Biomed News
on Therapy resistance biology in myeloid leukemia
Issue of 2021‒10‒24
twenty-nine papers selected by
Paolo Gallipoli
Barts Cancer Institute, Queen Mary University of London

  1. Cancers (Basel). 2021 Oct 19. pii: 5243. [Epub ahead of print]13(20):
      Relapses and resistance to therapeutic agents are major barriers in the treatment of acute myeloid leukemia (AML) patients. These unfavorable outcomes emphasize the need for new strategies targeting drug-resistant cells. As IDH mutations are present in the preleukemic stem cells and systematically conserved at relapse, targeting IDH mutant cells could be essential to achieve a long-term remission in the IDH mutant AML subgroup. Here, using a panel of human AML cell lines and primary AML patient specimens harboring IDH mutations, we showed that the production of an oncometabolite (R)-2-HG by IDH mutant enzymes induces vitamin D receptor-related transcriptional changes, priming these AML cells to differentiate with pharmacological doses of ATRA and/or VD. This activation occurs in a CEBPα-dependent manner. Accordingly, our findings illuminate potent and cooperative effects of IDH mutations and the vitamin D receptor pathway on differentiation in AML, revealing a novel therapeutic approach easily transferable/immediately applicable to this subgroup of AML patients.
    Keywords:  AML; ATRA; CEBPα; IDH; VDR; differentiation; vitamin D
  2. Cell Death Discov. 2021 Oct 18. 7(1): 297
      DNA (cytosine-5)-methyltransferase 3A (DNMT3A) mutations occur in ~20% of de novo acute myeloid leukemia (AML) patients, and >50% of these mutations in AML samples are heterozygous missense alterations within the methyltransferase domain at residue R882. DNMT3A R882 mutations in AML patients promote resistance to anthracycline chemotherapy and drive relapse. In this study, we performed high-throughput screening and identified that oridonin, an ent-kaurene diterpenoid extracted from the Chinese herb Rabdosia rubescens, inhibits DNMT3A R882 mutant leukemic cells at a low-micromolar concentration (IC50 = 2.1 µM) by activating both RIPK1-Caspase-8-Caspase-3-mediated apoptosis and RIPK1-RIPK3-MLKL-mediated necroptosis. The inhibitory effect of oridonin against DNMT3A R882 mutant leukemia cells can also be observed in vivo. Furthermore, oridonin inhibits clonal hematopoiesis of hematopoietic stem cells (HSCs) with Dnmt3a R878H mutation comparing to normal HSCs by inducing apoptosis and necroptosis. Overall, oridonin is a potential and promising drug candidate or lead compound targeting DNMT3A R882 mutation-driven clonal hematopoiesis and leukemia.
  3. Lancet Oncol. 2021 Oct 18. pii: S1470-2045(21)00494-0. [Epub ahead of print]
      BACKGROUND: Enasidenib is an oral inhibitor of mutant isocitrate dehydrogenase-2 (IDH2) proteins. We evaluated the safety and activity of enasidenib plus azacitidine versus azacitidine alone in patients with newly diagnosed, mutant-IDH2 acute myeloid leukaemia ineligible for intensive chemotherapy.METHODS: This open-label, phase 1b/2 trial was done at 43 clinical sites in 12 countries (the USA, Germany, Canada, the UK, France, Spain, Australia, Italy, the Netherlands, Portugal, Switzerland, and South Korea). Eligible patients were aged 18 years or older and had newly diagnosed, mutant-IDH2 acute myeloid leukaemia, and an Eastern Cooperative Oncology Group performance status of 0-2. In the phase 1b dose-finding portion, patients received oral enasidenib 100 mg/day or 200 mg/day in continuous 28-day cycles, plus subcutaneous azacitidine 75 mg/m2 per day for 7 days of each cycle. In phase 2, patients were randomly assigned (2:1) via an interactive web response system to enasidenib plus azacitidine or azacitidine-only, stratified by acute myeloid leukaemia subtype (de novo or secondary). The primary endpoint in the phase 2 portion was the overall response rate in the intention-to-treat population at a prespecified interim analysis (Aug 20, 2019) when all patients had at least 1 year of follow-up. Safety was assessed in all patients who received at least one dose of study drug. The trial is registered with, NCT02677922, and is ongoing.
    FINDINGS: Between June 3, 2016, and Aug 2, 2018, 322 patients were screened and 107 patients with mutant-IDH2 acute myeloid leukaemia were enrolled. At data cutoff for the interim analysis, 24 patients (including two from the phase 1 portion) were still receiving their assigned treatment. Six patients were enrolled in the phase 1b dose-finding portion of the trial and received enasidenib 100 mg (n=3) or 200 mg (n=3) in combination with azacitidine. No dose-limiting toxicities occurred and the enasidenib 100 mg dose was selected for phase 2. In phase 2, 101 patients were randomly assigned to enasidenib plus azacitidine (n=68) or azacitidine only (n=33). Median age was 75 years (IQR 71-78). 50 (74%; 95% CI 61-84) patients in the enasidenib plus azacitidine combination group and 12 (36%; 20-55) patients in the azacitidine monotherapy group achieved an overall response (odds ratio 4·9 [95% CI 2·0-11·9]; p=0·0003). Common treatment-related grade 3 or 4 adverse events with enasidenib plus azacitidine were thrombocytopenia (25 [37%] of 68 vs six [19%] of 32 in the azacitidine-only group), neutropenia (25 [37%] vs eight [25%]), anaemia (13 [19%] vs seven [22%]), and febrile neutropenia (11 [16%] vs five [16%]). Serious treatment-related adverse events were reported in 29 (43%) patients in the combination group and 14 (44%) patients in the azacitidine-only group; serious treatment-related adverse events occurring in more than 5% of patients in either group were febrile neutropenia (nine [13%] in the combination group vs five [16%] in the azacitidine-only group), differentiation syndrome (seven [10%] vs none), and pneumonia (three [4%] vs two [6%]). No treatment-related deaths were reported.
    INTERPRETATION: Combination enasidenib plus azacitidine was well tolerated and significantly improved overall response rates compared with azacitidine monotherapy, suggesting that this regimen can improve outcomes for patients with newly diagnosed, mutant-IDH2 acute myeloid leukaemia.
    FUNDING: Bristol Myers Squibb.
  4. Leukemia. 2021 Oct 20.
      Germline DDX41 variants in myeloid neoplasms (MNs) are not uncommon, and we explored the prevalence and characterized the clinical and pathologic features in a cohort of 3132 unrelated adult MN patients. By targeted next-generation sequencing, we identified 28 patients (20 men and 8 women) with pathogenic germline DDX41 variants who developed acute myeloid leukemia (AML), in which only 3 (11%) had a family history (FH) of MNs. A subacute clinical course of cytopenia (mean duration of 11.2 months, range 0-72 months) prior to the initial AML diagnosis was accompanied by a low blast count (median at 30%, range 20-70%) in hypocellular marrows (93% of all patients), in vast contrast to the typical proliferative subtypes of AML in the elderly. Most patients had a normal karyotype (75%) and acquired a second DDX41 variant (69%). A favorable overall survival (OS) was observed in comparison to that of common subtypes of AML with wild-type DDX41 in age-matched patients. Our study demonstrated that the frequent germline pathogenic DDX41 variants characterized a clinically distinct AML entity. Features characteristic of DDX41-mutated AML include male predominance, often lack of FH, indolent course, low proliferative potential, frequent somatic DDX41 variants, and a favorable OS.
  5. Am J Clin Pathol. 2021 Oct 19. pii: aqab172. [Epub ahead of print]
      OBJECTIVES: To explore the distinct mutation profiles between acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) and de novo AML and their relationships with prognosis.METHODS: Next-generation sequencing of 42 myeloid neoplasm-related genes in 293 newly diagnosed patients with AML.
    RESULTS: Eighty-four patients had AML-MRC, and 161 patients had de novo AML. The mutation rates of ASXL1 (25% vs 8.7%, P = .001), NRAS (17.9% vs 8.1%, P = .022), PTPN11 (11.9% vs 5%, P = .048), SETBP1 (6% vs 0.6%, P = .033), SRSF2 (11.9% vs 5.6%, P = .08), TP53 (16.7% vs 1.2%, P < .001), and U2AF1 (17.9% vs 7.5%, P = .014) in AML-MRC were higher than those in de novo AML, while the rates of FLT3-ITD (3.6% vs 15.5%, P = .005), KIT (0% vs 6.2%, P = .046), WT1 (3.6% vs 9.9%, P = .077), NPM1 (1.2% vs 21.7%, P < .001), and CEBPA (4.8% vs 24.2%, P < .001) mutation were lower. The appearance of ASXL1, TP53, U2AF1, SRSF2, and SETBP1 mutation could predict AML-MRC-like features in de novo AML, which was related to older age (60 vs 51 years, P = .001), low WBC counts (4.7 × 109/L vs 11.6 × 109/L, P = .001), and inferior outcomes (median overall survival, 15 months vs not reached, P = .003).
    CONCLUSIONS: The presence of AML-MRC-related mutations can reveal a subset of patients with de novo AML similar to patients with AML-MRC.
    Keywords:  AML-MRC; Acute myeloid leukemia; De novo AML; Genetic mutation
  6. Blood Adv. 2021 Oct 21. pii: bloodadvances.2021006064. [Epub ahead of print]
      Acute promyelocytic leukemia (APL) is a specific subtype of acute myeloid leukemia (AML) characterized by block of differentiation at the promyelocytic stage and the presence of PML-RARA fusion. In rare instances, RARA is fused with other partners in variant APL. More infrequently, non-RARA genes are rearranged in AML patients resembling APL. However, the underlying disease pathogenesis in these atypical cases is largely unknown. Here, we report the identification and characterization of a NUP98-JADE2 fusion in a pediatric AML patient showing APL-like morphology and immunophenotype. Mechanistically, we showed that NUP98-JADE2 could impair all-trans retinoic acid (ATRA)-mediated transcriptional control and myeloid differentiation. Intriguingly, NUP98-JADE2 was found to alter the subcellular distribution of wild-type JADE2, whose down-regulation similarly led to attenuated ATRA-induced responses and myeloid activation, suggesting that NUP98-JADE2 may mediate JADE2 inhibition. To our knowledge, this is the first report of a NUP98-non-RAR rearrangement identified in an AML patient mimicking APL. Our findings suggest JADE2 as a novel myeloid player involved in retinoic acid-induced differentiation. Despite lacking a rearranged RARA, our findings implicate that altered retinoic acid signaling by JADE2 disruption may underlie the APL-like features in our case, corroborating the importance of this signaling in APL pathogenesis.
  7. Nat Commun. 2021 Oct 22. 12(1): 6154
      Acute myeloid leukemia (AML) harboring inv(16)(p13q22) expresses high levels of miR-126. Here we show that the CBFB-MYH11 (CM) fusion gene upregulates miR-126 expression through aberrant miR-126 transcription and perturbed miR-126 biogenesis via the HDAC8/RAN-XPO5-RCC1 axis. Aberrant miR-126 upregulation promotes survival of leukemia-initiating progenitors and is critical for initiating and maintaining CM-driven AML. We show that miR-126 enhances MYC activity through the SPRED1/PLK2-ERK-MYC axis. Notably, genetic deletion of miR-126 significantly reduces AML rate and extends survival in CM knock-in mice. Therapeutic depletion of miR-126 with an anti-miR-126 (miRisten) inhibits AML cell survival, reduces leukemia burden and leukemia stem cell (LSC) activity in inv(16) AML murine and xenograft models. The combination of miRisten with chemotherapy further enhances the anti-leukemia and anti-LSC activity. Overall, this study provides molecular insights for the mechanism and impact of miR-126 dysregulation in leukemogenesis and highlights the potential of miR-126 depletion as a therapeutic approach for inv(16) AML.
  8. Clin Lymphoma Myeloma Leuk. 2021 Sep 11. pii: S2152-2650(21)02038-3. [Epub ahead of print]
      BACKGROUND: Core-binding factor acute myeloid leukemia (CBF AML) with recurrent genetic abnormalities inv(16)(p13.1q22) or t(16;16)(p13.1;q22)/ CBFB-MYH11 are usually prognostically favorable but heterogeneous group and additional abnormalities change their prognosis.MATERIALS AND METHODS: To evaluate the impact of a complex karyotype on CBF AML prognosis, we included 24 patients with a median age of 56.4 years (23.2-83.3) and a median number of abnormalities of 5 (4-13).
    RESULTS: Median follow-up was 110.4 months. Among patients with a primary clone, complete remission (CR) was reached in 66.7% of patients. 31.3% of patients experienced AML relapse with a median of 8.5 months. Median OS for transplanted patients was 80.7 versus 40.5 months for non-transplanted patients, excluding the 4 patients with early death. Among patients harboring AML with clonal evolution, CR was reached in 62.5% of patients. 50% of patients underwent allogeneic stem cell transplantation (ASCT). In these, median RFS was 19.3 versus 0 months in non-transplanted patients. Median OS seemed also longer in transplanted patients with 23.5 versus 2.95 months in non-transplanted.
    CONCLUSION: Use of new treatment and tailored strategy based on measurable residual disease monitoring may now improve these results. However, these data allow us to reconsider the good prognosis historically associated with CBF patients despite of karyotype and the place of ASCT in the strategy.
    Keywords:  AML; Allograft; CBF; Karyotype; Prognosis
  9. Sci Rep. 2021 Oct 20. 11(1): 20745
      FLT3-ITD mutations are detected in approximately 25% of newly diagnosed adult acute myeloid leukemia (AML) patients and confer an adverse prognosis. The FLT3-ITD allelic ratio has clear prognostic value. Nevertheless, there are numerous manuscripts with contradictory results regarding the prognostic relevance of the length and insertion site (IS) of the FLT3-ITD fragment. We aimed to assess the prognostic impact of these variables on the complete remission (CR) rates, overall survival (OS) and relapse-free survival (RFS) of AML patients with FLT3-ITDmutations. We studied the FLT3-ITD length of 362 adult AML patients included in the PETHEMA AML registry. We tried to validate the thresholds of ITD length previously published (i.e., 39 bp and 70 bp) in intensively treated AML patients (n = 161). We also analyzed the mutational profile of 118 FLT3-ITD AML patients with an NGS panel of 39 genes and correlated mutational status with the length and IS of ITD. The AUC of the ROC curve of the ITD length for OS prediction was 0.504, and no differences were found when applying any of the thresholds for OS, RFS or CR rate. Only four out of 106 patients had ITD IS in the TKD1 domain. Our results, alongside previous publications, confirm that FLT3-ITD length lacks prognostic value and clinical applicability.
  10. Nat Genet. 2021 Oct 18.
      Acute myeloid and lymphoid leukemias often harbor chromosomal translocations involving the KMT2A gene, encoding the KMT2A lysine methyltransferase (also known as mixed-lineage leukemia-1), and produce in-frame fusions of KMT2A to other chromatin-regulatory proteins. Here we map fusion-specific targets across the genome for diverse KMT2A oncofusion proteins in cell lines and patient samples. By modifying CUT&Tag chromatin profiling for full automation, we identify common and tumor-subtype-specific sites of aberrant chromatin regulation induced by KMT2A oncofusion proteins. A subset of KMT2A oncofusion-binding sites are marked by bivalent (H3K4me3 and H3K27me3) chromatin signatures, and single-cell CUT&Tag profiling reveals that these sites display cell-to-cell heterogeneity suggestive of lineage plasticity. In addition, we find that aberrant enrichment of H3K4me3 in gene bodies is sensitive to Menin inhibitors, demonstrating the utility of automated chromatin profiling for identifying therapeutic vulnerabilities. Thus, integration of automated and single-cell CUT&Tag can uncover epigenomic heterogeneity within patient samples and predict sensitivity to therapeutic agents.
  11. Nat Commun. 2021 Oct 18. 12(1): 6061
      Mutations in the epigenetic modifier TET2 are frequent in myeloid malignancies and clonal hematopoiesis of indeterminate potential (CHIP) and clonal cytopenia of undetermined significance (CCUS). Here, we investigate associations between TET2 mutations and DNA methylation in whole blood in 305 elderly twins, 15 patients with CCUS and 18 healthy controls. We find that TET2 mutations are associated with DNA hypermethylation at enhancer sites in whole blood in CHIP and in both granulocytes and mononuclear cells in CCUS. These hypermethylated sites are associated with leukocyte function and immune response and ETS-related and C/EBP-related transcription factor motifs. While the majority of TET2-associated hypermethylation sites are shared between CHIP and in AML, we find a set of AML-specific hypermethylated loci at active enhancer elements in hematopoietic stem cells. In summary, we show that TET2 mutations is associated with hypermethylated enhancers involved in myeloid differentiation in both CHIP, CCUS and AML patients.
  12. Br J Cancer. 2021 Oct 22.
      BACKGROUND: We previously demonstrated the in vitro killing of AML cells by the combination of the lipid-lowering agent bezafibrate (BEZ) and the contraceptive hormone medroxyprogesterone acetate (MPA). A phase II trial demonstrated in vivo safety and efficacy of BEZ and MPA (BaP) in elderly, relapsed/refractory AML and high-risk myelodysplastic syndrome (MDS) patients. However, we observed dose-limiting toxicities in a second trial that attempted to improve outcomes via escalation of BaP doses. Thus we sought to identify a third repurposed drug that potentiates activity of low dose BaP (BaP 0.1 mM).METHODS AND RESULTS: We demonstrate that addition of a commonly used anti-epileptic, valproic acid (VAL) to low dose BaP (BaP 0.1 mM)(VBaP) enhanced killing of AML cell lines/primary AML cells to levels similar to high dose BaP (BaP 0.5 mM). Similarly, addition of VAL to BaP 0.1 mM enhanced reactive oxygen species (ROS), lipid peroxidation and inhibition of de novo fatty acid synthesis. Overexpression of Nrf2 in K562 and KG1a completely inhibited ROS production and rescued cells from VAL/BaP 0.1 mM/VBaP killing.
    CONCLUSIONS: Given the good safety data of low-dose BaP in elderly/relapsed/refractory AML patients, and that VAL alone is well-tolerated, we propose VBaP as a novel therapeutic combination for AML.
  13. Blood Cancer Discov. 2020 Sep;1(2): 134-145
      Targeting Hedgehog (Hh) pathway components, such as Smoothened (SMO), is a developing strategy for the treatment of acute myeloid leukemia (AML) and for overcoming relapsed/refractory forms of this disease. Several SMO inhibitors are in clinical development for the treatment of various tumor types and the results from some clinical trials in AML have been reported. This review will discuss the role of Hh signaling in AML pathogenesis, describe the preclinical and clinical development of Hh pathway inhibitors for the treatment of AML, and examine the current evidence on Hh pathway inhibitor resistance and the implications for treatment selection in AML.Significance: In acute myeloid leukemia (AML), components of the Hedgehog (Hh) signaling pathway, such as Smoothened (SMO), have been implicated in the development, maintenance, and expansion of leukemic stem cells (LSC), as well as sensitization to chemotherapy and the development of drug resistance in AML. Observations in preclinical studies of AML, as well as from samples of patients with AML, demonstrate that Hh pathway inhibitors act primarily on the stem cell pathway as differentiation agents. The current data for hematologic malignancies indicate the potential for a synergistic effect when a Hh pathway inhibitor is administered in combination with chemotherapy or investigational agents. It is thought that Hh pathway inhibitors act as agents that reduce LSC dormancy and promote LSC differentiation, thus the newly dividing LSCs can then be targeted by other chemotherapeutic drugs.
  14. Nat Med. 2021 Oct 18.
      Clonal hematopoiesis (CH) results from somatic genomic alterations that drive clonal expansion of blood cells. Somatic gene mutations associated with hematologic malignancies detected in hematopoietic cells of healthy individuals, referred to as CH of indeterminate potential (CHIP), have been associated with myeloid malignancies, while mosaic chromosomal alterations (mCAs) have been associated with lymphoid malignancies. Here, we analyzed CHIP in 55,383 individuals and autosomal mCAs in 420,969 individuals with no history of hematologic malignancies in the UK Biobank and Mass General Brigham Biobank. We distinguished myeloid and lymphoid somatic gene mutations, as well as myeloid and lymphoid mCAs, and found both to be associated with risk of lineage-specific hematologic malignancies. Further, we performed an integrated analysis of somatic alterations with peripheral blood count parameters to stratify the risk of incident myeloid and lymphoid malignancies. These genetic alterations can be readily detected in clinical sequencing panels and used with blood count parameters to identify individuals at high risk of developing hematologic malignancies.
  15. Clin Lymphoma Myeloma Leuk. 2021 Sep 24. pii: S2152-2650(21)02044-9. [Epub ahead of print]
      INTRODUCTION/BACKGROUND: The AML-Score has been validated in patients receiving traditional induction chemotherapies but not CPX-351. We conducted a retrospective analysis to evaluate, among patients with secondary acute myeloid leukemia who received intensive induction with CPX-351, if the AML-Score associates with (1) complete remission (CR) and (2) early mortality (EM) within 60 days of induction.MATERIALS AND METHODS: We abstracted demographic and clinical data from consecutive patients receiving CPX-351 at Sidney Kimmel Cancer Center at Thomas Jefferson University Hospital between September 2017 and November 2020. We used descriptive statistics and receiver operating curves to evaluate the relationship between AML-Score and rates of CR and EM.
    RESULTS: In total, 40 patients were included. 27 (67.5%) were male, 27 (67.5%) were white, 36 (90.0%) were not Hispanic or Latino, and 29 (72.5%) were aged ≥60 years. Twenty-seven patients (67.5%) had a CR, and 4 (10%) experienced EM. Observed rates of CR and EM generally increased with increasing predicted risk. The area under the curve was 0.75 (95% CI 0.60-0.90) for CR and 0.82 (95% CI 0.68-0.96) for EM.
    CONCLUSION: The AML-Score tool trends in the correct direction for predicting CR and EM, and thus may facilitate oncologist prognostication and treatment planning for patients receiving CPX-351. However, its clinical utility is limited by its underestimation of the risk of CR and overestimation of the risk of EM. Further validation in a larger cohort is needed to calculate accurate point estimates of CR and EM risk in this population.
    Keywords:  AML-score; Acute myeloid leukemia; Mortality; Risk stratification; Vyxeos (CPX-351)
  16. Am J Cancer Res. 2021 ;11(9): 4470-4484
      Ponatinib is a tyrosine kinase inhibitor (TKI) directed against BCR-ABL1 which is successfully used in patients with BCR-ABL1 T315I+ chronic myeloid leukemia (CML). However, BCR-ABL1 compound mutations may develop during therapy in these patients and may lead to drug resistance. Asciminib is a novel drug capable of targeting most BCR-ABL1 mutant-forms, including BCR-ABL1T315I, but remains ineffective against most BCR-ABL1T315I+ compound mutation-bearing sub-clones. We demonstrate that asciminib synergizes with ponatinib in inducing growth-arrest and apoptosis in patient-derived CML cell lines and murine Ba/F3 cells harboring BCR-ABL1 T315I or T315I-including compound mutations. Asciminib and ponatinib also produced cooperative effects on CRKL phosphorylation in BCR-ABL1-transformed cells. The growth-inhibitory effects of the drug combination 'asciminib+ponatinib' was further enhanced by hydroxyurea (HU), a drug which has lately been described to suppresses the proliferation of BCR-ABL1 T315I+ CML cells. Cooperative drug effects were also observed in patient-derived CML cells. Most importantly, we were able to show that the combinations 'asciminib+ponatinib' and 'asciminib+ponatinib+HU' produce synergistic apoptosis-inducing effects in CD34+/CD38- CML stem cells obtained from patients with chronic phase CML or BCR-ABL1 T315I+ CML blast phase. Together, asciminib, ponatinib and HU synergize in producing anti-leukemic effects in multi-resistant CML cells, including cells harboring T315I+ BCR-ABL1 compound mutations and CML stem cells. The clinical efficacy of this TKI combination needs to be evaluated within the frame of upcoming clinical trials.
    Keywords:  BCR-ABL1 compound mutations; BCR-ABL1T315I; CML; asciminib; drug combinations; hydroxyurea; leukemic stem cells; ponatinib
  17. Leuk Res. 2021 Oct 15. pii: S0145-2126(21)01729-X. [Epub ahead of print]111 106728
      Ultra-deep sequencing detects low-frequency genetic mutations with high sensitivity. We used this approach to prospectively examine mutations in the BCR/ABL1 tyrosine kinase from patients with newly diagnosed, chronic-phase chronic myeloid leukemia (CML) treated with the tyrosine kinase inhibitor nilotinib. Between May 2013 and November 2014, 50 patients from 18 institutions were enrolled in the study. We screened 103 somatic mutations and found that mutations in the P-loop domain were the most frequent (173/454 mutations in the P-loop) and noted the presence of the V299 L mutation (dasatinib-resistant/nilotinib-sensitive) in 98 % of patients (49/50). No patients had Y253H, E255 V, or F359 V/C/I mutations, which would recommend dasatinib rather than nilotinib treatment. The S417Y mutation was associated with lower achievement of a major molecular response (MMR) at 6 months, and the V371A mutation was associated with reduced MMR and MR4.5 durations (MMR for 2 years: 100 % for no mutation vs. 75 % for mutation, P=0.039; MR4.5 for 15 months: 94.1 % vs. 25 %, P=0.002). Patients with known nilotinib-resistant mutations had lower rates of MR4.5 achievement. In conclusion, ultra-deep sequencing is a sensitive method for genetic-based treatment decisions. Based on the results of these mutational analyses, nilotinib treatment is a promising option for Korean patients with CML.
    Keywords:  BCR-ABL1 tyrosine kinase; Chronic myeloid leukemia; Molecular response; Mutations; Ultra-deep sequencing
  18. Blood Cancer Discov. 2021 May;2(3): 238-249
      In myelodysplastic syndrome (MDS) and myeloproliferative neoplasm (MPN), bone marrow (BM) histopathology is assessed to identify dysplastic cellular morphology, cellularity, and blast excess. Yet, other morphologic findings may elude the human eye. We used convolutional neural networks to extract morphologic features from 236 MDS, 87 MDS/MPN, and 11 control BM biopsies. These features predicted genetic and cytogenetic aberrations, prognosis, age, and gender in multivariate regression models. Highest prediction accuracy was found for TET2 [area under the receiver operating curve (AUROC) = 0.94] and spliceosome mutations (0.89) and chromosome 7 monosomy (0.89). Mutation prediction probability correlated with variant allele frequency and number of affected genes per pathway, demonstrating the algorithms' ability to identify relevant morphologic patterns. By converting regression models to texture and cellular composition, we reproduced the classical del(5q) MDS morphology consisting of hypolobulated megakaryocytes. In summary, this study highlights the potential of linking deep BM histopathology with genetics and clinical variables.Significance: Histopathology is elementary in the diagnostics of patients with MDS, but its high-dimensional data are underused. By elucidating the association of morphologic features with clinical variables and molecular genetics, this study highlights the vast potential of convolutional neural networks in understanding MDS pathology and how genetics is reflected in BM morphology. See related commentary by Elemento, p. 195.
  19. Bone Marrow Transplant. 2021 Oct 20.
      The mutant burden of FLT3-ITD modulates its prognostic impact on patients with acute myeloid leukemia (AML). However, for patients with low allelic ratio (AR) FLT3-ITD (FLT3-ITDlow, AR < 0.5), clinical features, as well as genomic and transcriptomic profiles remain unclear, and evidence supporting allogeneic hematopoietic stem cell transplantation (allo-HSCT) in first complete remission (CR1) remains controversial. This study aimed to elucidate the genomic features, prognosis, and transplantation outcome of FLT3-ITDIow in AML patients with intermediate-risk cytogenetics. FLT3-ITDlow was associated with a negative enrichment of the leukemic stem cell signature, a marked enrichment of the RAS pathway, and with higher frequencies of RAS pathway mutations, different from those with FLT3-ITDhigh. Concurrent CEBPA double mutations were favorable prognostic factors, whereas MLL-PTD, and mutations in splicing factors were unfavorable prognostic factors in FLT3-ITDlow patients. Patients with FLT3-ITDlow had a shorter overall survival (OS) and event-free survival (EFS) than those with FLT3wt. Allo-HSCT in CR1 was associated with a significantly longer OS and EFS compared with postremission chemotherapy in patients with FLT3-ITDlow. In conclusion, FLT3-ITDlow is associated with different mutational and transcriptomic profiles compared with FLT3-ITDhigh. The presence of concomitant poor-risk mutations exert negative prognostic impacts in patients with FLT3-ITDlow, who markedly benefit from allo-HSCT in CR1.
  20. Stem Cell Reports. 2021 Oct 09. pii: S2213-6711(21)00496-3. [Epub ahead of print]
      Calreticulin (CALR) mutations are driver mutations in myeloproliferative neoplasms (MPNs), leading to activation of the thrombopoietin receptor and causing abnormal megakaryopoiesis. Here, we generated patient-derived CALRins5- or CALRdel52-positive induced pluripotent stem cells (iPSCs) to establish an MPN disease model for molecular and mechanistic studies. We demonstrated myeloperoxidase deficiency in granulocytic cells derived from homozygous CALR mutant iPSCs, rescued by repairing the mutation using CRISPR/Cas9. iPSC-derived megakaryocytes showed characteristics of primary megakaryocytes such as formation of demarcation membrane system and cytoplasmic pro-platelet protrusions. Importantly, CALR mutations led to enhanced megakaryopoiesis and accelerated megakaryocytic development in a thrombopoietin-independent manner. Mechanistically, our study identified differentially regulated pathways in mutated versus unmutated megakaryocytes, such as hypoxia signaling, which represents a potential target for therapeutic intervention. Altogether, we demonstrate key aspects of mutated CALR-driven pathogenesis dependent on its zygosity, and found novel therapeutic targets, making our model a valuable tool for clinical drug screening in MPNs.
    Keywords:  MPN; RNA sequencing; calreticulin mutation; essential thrombocythemia; iPSCs; induced pluripotent stem cells; megakaryocytes; myelofibrosis; myeloproliferative neoplasm; transcriptional profiling
  21. Nat Commun. 2021 Oct 18. 12(1): 6071
      In contrast to the curative effect of allogenic stem cell transplantation in acute myeloid leukemia via T cell activity, only modest responses are achieved with checkpoint-blockade therapy, which might be explained by T cell phenotypes and T cell receptor (TCR) repertoires. Here, we show by paired single-cell RNA analysis and TCR repertoire profiling of bone marrow cells in relapsed/refractory acute myeloid leukemia patients pre/post azacytidine+nivolumab treatment that the disease-related T cell subsets are highly heterogeneous, and their abundance changes following PD-1 blockade-based treatment. TCR repertoires expand and primarily emerge from CD8+ cells in patients responding to treatment or having a stable disease, while TCR repertoires contract in therapy-resistant patients. Trajectory analysis reveals a continuum of CD8+ T cell phenotypes, characterized by differential expression of granzyme B and a bone marrow-residing memory CD8+ T cell subset, in which a population with stem-like properties expressing granzyme K is enriched in responders. Chromosome 7/7q loss, on the other hand, is a cancer-intrinsic genomic marker of PD-1 blockade resistance in AML. In summary, our study reveals that adaptive T cell plasticity and genomic alterations determine responses to PD-1 blockade in acute myeloid leukemia.
  22. Nat Commun. 2021 Oct 18. 12(1): 6060
      The nucleotide analogue azacitidine (AZA) is currently the best treatment option for patients with high-risk myelodysplastic syndromes (MDS). However, only half of treated patients respond and of these almost all eventually relapse. New treatment options are urgently needed to improve the clinical management of these patients. Here, we perform a loss-of-function shRNA screen and identify the histone acetyl transferase and transcriptional co-activator, CREB binding protein (CBP), as a major regulator of AZA sensitivity. Compounds inhibiting the activity of CBP and the closely related p300 synergistically reduce viability of MDS-derived AML cell lines when combined with AZA. Importantly, this effect is specific for the RNA-dependent functions of AZA and not observed with the related compound decitabine that is only incorporated into DNA. The identification of immediate target genes leads us to the unexpected finding that the effect of CBP/p300 inhibition is mediated by globally down regulating protein synthesis.
  23. Leukemia. 2021 Oct 16.
      Chromosomal abnormalities are established prognostic markers in adult ALL. We assessed the prognostic impact of established chromosomal abnormalities and key copy number alterations (CNA) among 652 patients with B-cell precursor ALL treated on a modern MRD driven protocol. Patients with KMT2A-AFF1, complex karyotype (CK) and low hypodiploidy/near-triploidy (HoTr) had high relapse rates 50%, 60% & 53% and correspondingly poor survival. Patients with BCR-ABL1 had an outcome similar to other patients. JAK-STAT abnormalities (CRLF2, JAK2) occurred in 6% patients and were associated with a high relapse rate (56%). Patients with ABL-class fusions were rare (1%). A small group of patients with ZNF384 fusions (n = 12) had very good survival. CNA affecting IKZF1, CDKN2A/B, PAX5, BTG1, ETV6, EBF1, RB1 and PAR1 were assessed in 436 patients. None of the individual deletions or profiles were associated with survival, either in the cohort overall or within key subgroups. Collectively these data indicate that primary genetic abnormalities are stronger prognostic markers than secondary deletions. We propose a revised UKALL genetic risk classification based on key established chromosomal abnormalities: (1) very high risk: CK, HoTr or JAK-STAT abnormalities; (2) high risk: KMT2A fusions; (3) Tyrosine kinase activating: BCR-ABL1 and ABL-class fusions; (4) standard risk: all other patients.
  24. Blood. 2021 Oct 17. pii: blood.2021013054. [Epub ahead of print]
      The key immunologic signatures associated with clinical outcomes after post-transplant cyclophosphamide (PTCy)-based HLA-haploidentical (haplo) and HLA-matched bone marrow transplantation (BMT) are largely unknown. To address this gap in knowledge, we used machine learning to decipher clinically relevant signatures from immunophenotypic, proteomic, and clinical data and then examined transcriptome changes in the lymphocyte subsets that predicted major post-transplant outcomes. Kinetics of immune subset reconstitution after day 28 were similar for 70 patients undergoing haplo and 75 patients undergoing HLA-matched BMT. Machine learning based on 35 candidate factors (10 clinical, 18 cellular, and 7 proteomic) revealed that combined elevations in effector CD4+ conventional T cells (Tconv) and CXCL9 at day 28 predicted acute graft-versus-host disease (aGVHD). Furthermore, higher NK cell counts predicted improved overall survival due to a reduction in both nonrelapse mortality and relapse. Transcriptional and flow-cytometric analyses of recovering lymphocytes in patients with aGVHD identified preserved hallmarks of functional CD4+ regulatory T cells (Tregs) while highlighting a Tconv-driven inflammatory and metabolic axis distinct from that seen with conventional GVHD prophylaxis. Patients developing early relapse displayed a loss of inflammatory gene signatures in NK cells and a transcriptional exhaustion phenotype in CD8+ T cells. Using a multimodality approach, we highlight the utility of systems biology in BMT biomarker discovery and offer a novel understanding of how PTCy influences alloimmune responses. Our work charts future directions for novel therapeutic interventions after these increasingly utilized GVHD prophylaxis platforms.
  25. Blood. 2021 Oct 17. pii: blood.2021011775. [Epub ahead of print]
      The cellular mechanisms required to ensure homeostasis of the hematopoietic niche and the ability of this niche to support hematopoiesis upon stress remain elusive. We here identify Wnt5a in Osterix+ mesenchymal progenitor and stem cells (MSPCs) as a critical factor for niche-dependent hematopoiesis. Mice lacking Wnt5a in MSPCs suffer from stress-related bone marrow failure and increased mortality. Niche cells devoid of Wnt5a show defective actin stress fiber orientation due to an elevated activity of the small Rho GTPase CDC42. This results in incorrect positioning of autophagosomes and lysosomes, thus reducing autophagy and increasing oxidative stress. In MSPCs from patients from bone marrow (BM) failure states which share features of peripheral cytopenia and hypocellular BM, we find similar defects in actin stress fiber orientation, reduced and incorrect colocalization of autophagosomes and lysosomes, and CDC42 activation. Strikingly, a short pharmacological intervention to attenuate elevated CDC42 activation in vivo in mice prevents defective actin- anchored autophagy in MSPCs, salvages hematopoiesis and protects against lethal cytopenia upon stress. In summary, our study identifies Wnt5a as a restriction factor for niche homeostasis by affecting CDC42-regulated actin stress-fiber orientation and autophagy upon stress. Our data further imply a critical role for autophagy in MSPCs for adequate support of hematopoiesis by the niche upon stress and in human diseases characterized by peripheral cytopenias and hypocellular BM.
  26. Br J Haematol. 2021 Oct 18.
      Over the last decade, the field of hereditary haematological malignancy syndromes (HHMSs) has gained increasing recognition among clinicians and scientists worldwide. Germline mutations now account for almost 10% of adult and paediatric myelodysplasia/acute myeloid leukaemia (MDS/AML). As our ability to diagnose HHMSs has improved, we are now faced with the challenges of integrating these advances into routine clinical practice for patients with MDS/AML and how to optimise management and surveillance of patients and asymptomatic carriers. Discoveries of novel syndromes combined with clinical, genetic and epigenetic profiling of tumour samples, have highlighted unique patterns of disease evolution across HHMSs. Despite these advances, causative lesions are detected in less than half of familial cases and evidence-based guidelines are often lacking, suggesting there is much still to learn. Future research efforts are needed to sustain current momentum within the field, led not only by advancing genetic technology but essential collaboration between clinical and academic communities.
    Keywords:  acute myeloid leukaemia; familial; germline; malignancy; testing
  27. Cell Stem Cell. 2021 Oct 13. pii: S1934-5909(21)00390-8. [Epub ahead of print]
      N6-methyladenosine (m6A) on mRNA plays critical roles in various cellular processes. However, the landscape and dynamics of m6A modification in hematopoietic system remain unknown. Here, we delineate a comprehensive m6A landscape across hematopoietic hierarchy and uncover that IGF2BP2 is required for preserving the function of hematopoietic stem cells (HSCs). Our data reveal a cell-type-specific m6A landscape in hematopoiesis. m6A modifications arise mostly in the early stage of hematopoiesis and prefer to play distinct roles for determining mRNA fates in HSCs and committed progenitors. Mechanistically, increased m6A-IGF2BP2 expression controls transcriptional state and maintenance of HSCs. IGF2BP2 deficiency induces quiescence loss and impairs HSC function. Moreover, IGF2BP2 loss increases mitochondrial activity of HSCs by accelerating Bmi1 mRNA decay, leading to de-repression of mitochondria-related genes. Collectively, our results present a fascinating portrait of m6A modification of hematopoietic hierarchy and reveal a key role of IGF2BP2 in maintaining HSC function by restraining mitochondrial activity.
    Keywords:  IGF2BP2; N6-methyladenosine; hematopoiesis; hematopoietic stem cells