bims-tremyl Biomed News
on Therapy resistance biology in myeloid leukemia
Issue of 2021‒07‒25
thirty-two papers selected by
Paolo Gallipoli
Barts Cancer Institute, Queen Mary University of London

  1. Blood. 2021 Jul 22. 138(3): 234-245
      Venetoclax, a Bcl-2 inhibitor, in combination with the hypomethylating agent azacytidine, achieves complete remission with or without count recovery in ∼70% of treatment-naive elderly patients unfit for conventional intensive chemotherapy. However, the mechanism of action of this drug combination is not fully understood. We discovered that venetoclax directly activated T cells to increase their cytotoxicity against acute myeloid leukemia (AML) in vitro and in vivo. Venetoclax enhanced T-cell effector function by increasing reactive oxygen species generation through inhibition of respiratory chain supercomplexes formation. In addition, azacytidine induced a viral mimicry response in AML cells by activating the STING/cGAS pathway, thereby rendering the AML cells more susceptible to T cell-mediated cytotoxicity. Similar findings were seen in patients treated with venetoclax, as this treatment increased reactive oxygen species generation and activated T cells. Collectively, this study presents a new immune-mediated mechanism of action for venetoclax and azacytidine in the treatment of AML and highlights a potential combination of venetoclax and adoptive cell therapy for patients with AML.
  2. Cell Stem Cell. 2021 Jul 12. pii: S1934-5909(21)00274-5. [Epub ahead of print]
      Current treatments for acute myeloid leukemia (AML) are often ineffective in eliminating leukemic stem cells (LSCs), which perpetuate the disease. Here, we performed a metabolic drug screen to identify LSC-specific vulnerabilities and found that nicotinamide phosphoribosyltransferase (NAMPT) inhibitors selectively killed LSCs, while sparing normal hematopoietic stem and progenitor cells. Treatment with KPT-9274, a NAMPT inhibitor, suppressed the conversion of saturated fatty acids to monounsaturated fatty acids, a reaction catalyzed by the stearoyl-CoA desaturase (SCD) enzyme, resulting in apoptosis of AML cells. Transcriptomic analysis of LSCs treated with KPT-9274 revealed an upregulation of sterol regulatory-element binding protein (SREBP)-regulated genes, including SCD, which conferred partial protection against NAMPT inhibitors. Inhibition of SREBP signaling with dipyridamole enhanced the cytotoxicity of KPT-9274 on LSCs in vivo. Our work demonstrates that altered lipid homeostasis plays a key role in NAMPT inhibitor-induced apoptosis and identifies NAMPT inhibition as a therapeutic strategy for targeting LSCs in AML.
    Keywords:  NAD metabolism; NAMPT; SREBP signaling; acute myeloid leukemia; dipyridamole; drug screen; fatty acid; leukemic stem cells; lipotoxicity; metabolism
  3. Cancer Discov. 2021 Jul 23. pii: candisc.0177.2021. [Epub ahead of print]
      Acute myeloid leukemia (AML) pathogenesis often involves a mutation in the NPM1 nucleolar chaperone, but the bases for its transforming properties and overall association with favorable therapeutic responses remain incompletely understood. Here we demonstrate that an oncogenic mutant form of NPM1 (NPM1c) impairs mitochondrial function. NPM1c also hampers formation of PML nuclear bodies (NBs), which are regulators of mitochondrial fitness and key senescence effectors. Actinomycin D (ActD), an antibiotic with unambiguous clinical efficacy in relapsed/refractory NPM1c-AMLs, targets these primed mitochondria, releasing mtDNA, activating cGAS signaling and boosting ROS production. The latter restore PML NB formation to drive TP53 activation and senescence of NPM1c-AML cells. In several models, dual targeting of mitochondria by venetoclax and ActD synergized to clear AML and prolong survival through targeting of PML. Our studies reveal an unexpected role for mitochondria downstream of NPM1c and implicate a mitochondrial/ROS/PML/TP53 senescence pathway as an effector of ActD-based therapies.
  4. Am J Hematol. 2021 Jul 21.
      SRSF2 mutations are frequently found in acute myeloid leukemia (AML) and mostly affect the P95 residue. Mutation in this splicing factor mediate abnormal splicing associated with exon skipping events, including EZH2 as a crucial target. While SRSF2 mutations are enriched in secondary AML and associated with worse outcomes following chemotherapy consolidation, very little is known about the associated biological and clinical implications in AML patients consolidated with allogeneic hematopoietic stemcell transplantation (HSCT). Here we retrospectively analyzed 263 adult AML patients who received an allogeneic HSCT regarding the biological and clinical implications of the SRSF2 mutation status at diagnosis and in morphologic remission at HSCT. We found 12.5% of the patients to be SRSF2 mutated at diagnosis. Mutated patients had increased EZH2 missplicing events with P95H likely driving this pathobiology most effectively. However, the amount of EZH2 missplicing events, as a functional surrogate marker did not associate with relevant biological or clinical characteristics. We observed a persistence of mutations in remission before HSCT in the majority (93%) of SRSF2 mutated AML patients. Importantly, the variant allele frequency (VAF) levels of SRSF2 mutations in remission at HSCT did not correlate with outcomes following HSCT consolidation, limiting the applicability of SRSF2 mutations as a marker for residual AML disease. Following allogeneic HSCT SRSF2 mutated AML patients experienced a 2-year overall survival of 77%, indicating that SRSF2 mutated AML patients may benefit from HSCT consolidation. This article is protected by copyright. All rights reserved.
  5. Sci Adv. 2021 Jul;pii: eabf3895. [Epub ahead of print]7(30):
      Metabolic dysregulation underlies malignant phenotypes attributed to cancer stem cells, such as unlimited proliferation and differentiation blockade. Here, we demonstrate that NAD+ metabolism enables acute myeloid leukemia (AML) to evade apoptosis, another hallmark of cancer stem cells. We integrated whole-genome CRISPR screening and pan-cancer genetic dependency mapping to identify NAMPT and NMNAT1 as AML dependencies governing NAD+ biosynthesis. While both NAMPT and NMNAT1 were required for AML, the presence of NAD+ precursors bypassed the dependence of AML on NAMPT but not NMNAT1, pointing to NMNAT1 as a gatekeeper of NAD+ biosynthesis. Deletion of NMNAT1 reduced nuclear NAD+, activated p53, and increased venetoclax sensitivity. Conversely, increased NAD+ biosynthesis promoted venetoclax resistance. Unlike leukemia stem cells (LSCs) in both murine and human AML xenograft models, NMNAT1 was dispensable for hematopoietic stem cells and hematopoiesis. Our findings identify NMNAT1 as a previously unidentified therapeutic target that maintains NAD+ for AML progression and chemoresistance.
  6. NPJ Precis Oncol. 2021 Jul 23. 5(1): 71
      The FDA recently approved eight targeted therapies for acute myeloid leukemia (AML), including the BCL-2 inhibitor venetoclax. Maximizing efficacy of these treatments requires refining patient selection. To this end, we analyzed two recent AML studies profiling the gene expression and ex vivo drug response of primary patient samples. We find that ex vivo samples often exhibit a general sensitivity to (any) drug exposure, independent of drug target. We observe that this "general response across drugs" (GRD) is associated with FLT3-ITD mutations, clinical response to standard induction chemotherapy, and overall survival. Further, incorporating GRD into expression-based regression models trained on one of the studies improved their performance in predicting ex vivo response in the second study, thus signifying its relevance to precision oncology efforts. We find that venetoclax response is independent of GRD but instead show that it is linked to expression of monocyte-associated genes by developing and applying a multi-source Bayesian regression approach. The method shares information across studies to robustly identify biomarkers of drug response and is broadly applicable in integrative analyses.
  7. Mol Cell. 2021 Jul 12. pii: S1097-2765(21)00507-4. [Epub ahead of print]
      Mutant isocitrate dehydrogenase (IDH) 1 and 2 play a pathogenic role in cancers, including acute myeloid leukemia (AML), by producing oncometabolite 2-hydroxyglutarate (2-HG). We recently reported that tyrosine phosphorylation activates IDH1 R132H mutant in AML cells. Here, we show that mutant IDH2 (mIDH2) R140Q commonly has K413 acetylation, which negatively regulates mIDH2 activity in human AML cells by attenuating dimerization and blocking binding of substrate (α-ketoglutarate) and cofactor (NADPH). Mechanistically, K413 acetylation of mitochondrial mIDH2 is achieved through a series of hierarchical phosphorylation events mediated by tyrosine kinase FLT3, which phosphorylates mIDH2 to recruit upstream mitochondrial acetyltransferase ACAT1 and simultaneously activates ACAT1 and inhibits upstream mitochondrial deacetylase SIRT3 through tyrosine phosphorylation. Moreover, we found that the intrinsic enzyme activity of mIDH2 is much higher than mIDH1, thus the inhibitory K413 acetylation optimizes leukemogenic ability of mIDH2 in AML cells by both producing sufficient 2-HG for transformation and avoiding cytotoxic accumulation of intracellular 2-HG.
    Keywords:  2-HG; ACAT1; AML; FLT3; K413 acetylation; SIRT3; dimerization; mutant IDH2
  8. Int J Mol Sci. 2021 Jul 16. pii: 7602. [Epub ahead of print]22(14):
      FMS-like tyrosine kinase 3 (FLT3) gene mutations have been found in more than one-third of Acute Myeloid Leukemia (AML) cases. The most common point mutation in FLT3 occurs at the 835th residue (D835A/E/F/G/H/I/N/V/Y), in the activation loop region. The D835 residue is critical in maintaining FLT3 inactive conformation; these mutations might influence the interaction with clinically approved AML inhibitors used to treat the AML. The molecular mechanism of each of these mutations and their interactions with AML inhibitors at the atomic level is still unknown. In this manuscript, we have investigated the structural consequence of native and mutant FLT-3 proteins and their molecular mechanisms at the atomic level, using molecular dynamics simulations (MDS). In addition, we use the molecular docking method to investigate the binding pattern between the FLT-3 protein and AML inhibitors upon mutations. This study apparently elucidates that, due to mutations in the D835, the FLT-3 structure loses its conformation and becomes more flexible compared to the native FLT3 protein. These structural changes are suggested to contribute to the relapse and resistance responses to AML inhibitors. Identifying the effects of FLT3 at the molecular level will aid in developing a personalized therapeutic strategy for treating patients with FLT-3-associated AML.
    Keywords:  AML; FLT3; flexibility; inhibitors; interaction; leukemia; mutations
  9. Cancers (Basel). 2021 Jul 10. pii: 3464. [Epub ahead of print]13(14):
      The ribonucleotide reductase inhibitor hydroxyurea suppresses de novo dNTP synthesis and attenuates the hyperproliferation of leukemic blasts. Mechanisms that determine whether cells undergo apoptosis in response to hydroxyurea are ill-defined. We used unbiased proteomics to uncover which pathways control the transition of the hydroxyurea-induced replication stress into an apoptotic program in chronic and acute myeloid leukemia cells. We noted a decrease in the serine/threonine kinase RAF1/c-RAF in cells that undergo apoptosis in response to clinically relevant doses of hydroxyurea. Using the RAF inhibitor LY3009120, we show that RAF activity determines the sensitivity of leukemic cells toward hydroxyurea. We further disclose that pharmacological inhibition of the RAF downstream target BCL-XL with the drug navitoclax and RNAi combine favorably with hydroxyurea against leukemic cells. BCR-ABL1 and hyperactive FLT3 are tyrosine kinases that causally contribute to the development of leukemia and induce RAF1 and BCL-XL. Accordingly, the ABL inhibitor imatinib and the FLT3 inhibitor quizartinib sensitize leukemic cells to pro-apoptotic effects of hydroxyurea. Moreover, hydroxyurea and navitoclax kill leukemic cells with mutant FLT3 that are resistant to quizartinib. These data reveal cellular susceptibility factors toward hydroxyurea and how they can be exploited to eliminate difficult-to-treat leukemic cells with clinically relevant drug combinations.
    Keywords:  AML; BCL-XL; BCR-ABL1; CML; FLT3; RAF1; apoptosis; hydroxyurea; replication stress
  10. Cancers (Basel). 2021 Jul 13. pii: 3499. [Epub ahead of print]13(14):
      Oxidative metabolism is crucial for leukemic stem cell (LSC) function and drug resistance in acute myeloid leukemia (AML). Mitochondrial metabolism also affects the immune system and therefore the anti-tumor response. The modulation of oxidative phosphorylation (OxPHOS) has emerged as a promising approach to improve the therapy outcome for AML patients. However, the effect of mitochondrial inhibitors on the immune compartment in the context of AML is yet to be explored. Immune checkpoints such as ectonucleotidase CD39 and programmed dead ligand 1 (PD-L1) have been reported to be expressed in AML and linked to chemo-resistance and a poor prognosis. In the present study, we first demonstrated that a novel selective electron transfer chain complex (ETC) I inhibitor, EVT-701, decreased the OxPHOS metabolism of murine and human cytarabine (AraC)-resistant leukemic cell lines. Furthermore, we showed that while AraC induced an immune response regulation by increasing CD39 expression and by reinforcing the interferon-γ/PD-L1 axis, EVT-701 reduced CD39 and PD-L1 expression in vitro in a panel of both murine and human AML cell lines, especially upon AraC treatment. Altogether, this work uncovers a non-canonical function of ETCI in controlling CD39 and PD-L1 immune checkpoints, thereby improving the anti-tumor response in AML.
    Keywords:  AML; Immune checkpoints; OxPHOS
  11. Leukemia. 2021 Jul 23.
      In spite of the remarkable progress in basic and preclinical studies of acute myeloid leukemia (AML), the five-year survival rate of AML patients remains poor, highlighting the urgent need for novel and synergistic therapies. Over the past decade, increased attention has been focused on identifying suitable immunotherapeutic strategies for AML, and in particular on targeting leukemic cells and their progenitors. However, recent studies have also underlined the important contribution of the leukemic microenvironment in facilitating tumor escape mechanisms leading to disease recurrence. Here, we describe the immunological features of the AML niche, with particular attention to the crosstalk between the AML blasts and the cellular components of the altered tumor microenvironment (TME) and the mechanisms of immune escape that hamper the therapeutic effects of the most advanced treatments. Considering the AML complexity, immunotherapy approaches may benefit from a rational combination of complementary strategies aimed at preventing escape mechanisms without increasing toxicity.
  12. Oncoimmunology. 2021 ;10(1): 1945804
      Chimeric antigen receptor T cells (CAR-T) targeting CD19 have achieved significant success in patients with B cell malignancies. To date, implementation of CAR-T in other indications remains challenging due to the lack of truly tumor-specific antigens as well as control of CAR-T activity in patients. CD123 is highly expressed in acute myeloid leukemia (AML) blasts including leukemia-initiating cells making it an attractive immunotherapeutic target. However, CD123 expression in normal hematopoietic progenitor cells and endothelia bears the risk of severe toxicities and may limit CAR-T applications lacking fine-tuned control mechanisms. Therefore, we recently developed a rapidly switchable universal CAR-T platform (UniCAR), in which CAR-T activity depends on the presence of a soluble adapter called targeting module (TM), and confirmed clinical proof-of-concept for targeting CD123 in AML with improved safety. As costimulation via 4-1BB ligand (4-1BBL) can enhance CAR-T expansion, persistence, and effector functions, a novel CD123-specific TM variant (TM123-4-1BBL) comprising trimeric single-chain 4-1BBL was developed for transient costimulation of UniCAR-T cells (UniCAR-T) at the leukemic site in trans. TM123-4-1BBL-directed UniCAR-T efficiently eradicated CD123-positive AML cells in vitro and in a CDX in vivo model. Moreover, additional costimulation via TM123-4-1BBL enabled enhanced expansion and persistence with a modulated UniCAR-T phenotype. In addition, the increased hydrodynamic volume of TM123-4-1BBL prolonged terminal plasma half-life and ensured a high total drug exposure in vivo. In conclusion, expanding the soluble adapter optionality for CD123-directed UniCAR-T maintains the platforms high anti-leukemic efficacy and immediate control mechanism for a flexible, safe, and individualized CAR-T therapy of AML patients.
    Keywords:  4-1BB; CD123; Chimeric antigen receptor (CAR); acute myeloid leukemia (AML); costimulation in trans; switchable CAR platform
  13. Cancer. 2021 Aug 15. 127(16): 2943-2953
      BACKGROUND: Acute myeloid leukemia (AML) is a heterogenous malignancy driven by genetic and epigenetic factors. Inhibition of bromodomain and extraterminal (BET) proteins, epigenetic readers that play pivotal roles in the regulation of genes relevant to cancer pathogenesis, constitutes a novel AML treatment approach.METHODS: In this first-in-human study of the pan-BET inhibitor mivebresib as monotherapy (MIV-mono) or in combination with venetoclax (MIV-Ven), the safety profile, efficacy, and pharmacodynamics of mivebresib were determined in patients with relapsed/refractory AML ( identifier NCT02391480). Mivebresib was administered at 3 monotherapy dose levels (1.5, 2.0, or 2.5 mg) or in combination with venetoclax (400 or 800 mg).
    RESULTS: Forty-four patients started treatment: of 19 who started MIV-mono, 5 went on to receive MIV-Ven combination therapy after disease progression and a washout period. Twenty-five patients started MIV-Ven, resulting in a total of 30 patients treated with the combination. The most common mivebresib-related treatment-emergent adverse events were dysgeusia (74%), decreased appetite (42%), and diarrhea (42%) in the MIV-mono group and decreased appetite (44%), vomiting (44%), and nausea (40%) in the MIV-Ven group. Serious adverse events occurred in 14 patients (74%) who received MIV-mono and in 22 patients (88%) who received MIV-Ven. In the MIV-mono group, responses were complete remission with incomplete blood count recovery in 1 patient and resistant disease in 15 patients. In the MIV-Ven group, responses were complete remission in 2 patients, partial remission in 2 patients, morphologic leukemia-free state in 2 patients, resistant disease in 12 patients, and aplasia in 1 patient. The pharmacodynamic effects of mivebresib were proportional to dose and drug exposure.
    CONCLUSIONS: Mivebresib was tolerated and showed antileukemic effects as monotherapy and in combination with venetoclax in patients with relapsed/refractory AML.
    LAY SUMMARY: Mivebresib is a novel drug that influences the way cancer cells read genetic information. Mivebresib was tested together with venetoclax in patients with acute myeloid leukemia after standard medicines failed and the disease returned, or when standard medicine was unavailable. Adverse effects were described for different drug doses, and the dose that is tolerable was determined. In some patients, their leukemia improved for some time. More studies are necessary to determine whether mivebresib can be used to treat acute myeloid leukemia.
    Keywords:  ABBV-075; acute myeloid leukemia; bromodomain and extraterminal domain protein; mivebresib; phase 1 clinical trial
  14. Cell Cycle. 2021 Jul 21. 1-9
      WT1 has been reported to function as an oncogene and a tumor suppressor in acute myeloid leukemia (AML). The molecular mechanisms have not yet been fully elucidated. Here, we report that p53, served as a tumor suppressor, plays a critical role in regulating the function of WT1 in AML. For details, we performed a meta-analysis on 1131 AML cases, showing that WT1 gene mutation and TP53 gene exhibited a mutually exclusive predisposition in AML. p53 can be recruited to the promoter region of WT1's target genes to modulate their expression by physically interacting with WT1. The AML-derived p53 mutation (p53R248Q) can disrupt the interaction between WT1 and p53, resulting in the loss of modulation of WT1's target genes. Furthermore, wild-type p53 maintained the anti-proliferation activity of WT1 in AML cells. In contrast, WT1 promoted AML cell proliferation in the absence of p53 (or mutated p53). In conclusion, we demonstrated a novel explanation of the controversial function of WT1 in AML. These results provided a mechanism by which WT1 inhibited AML cell proliferation in a p53-dependent manner.
    Keywords:  AML; WT1; p53; proliferation
  15. Cancers (Basel). 2021 Jul 10. pii: 3457. [Epub ahead of print]13(14):
      Nucleophosmin (NPM1) is a nucleocytoplasmic shuttling protein, predominantly located in the nucleolus, that regulates a multiplicity of different biological processes. NPM1 localization in the cell is finely tuned by specific signal motifs, with two tryptophan residues (Trp) being essential for the nucleolar localization. In acute myeloid leukemia (AML), several NPM1 mutations have been reported, all resulting in cytoplasmic delocalization, but the putative biological and clinical significance of different variants are still debated. We explored HOXA and HOXB gene expression profile in AML patients and found a differential expression between NPM1 mutations inducing the loss of two (A-like) Trp residues and those determining the loss of one Trp residue (non-A-like). We thus expressed NPM1 A-like- or non-A-like-mutated vectors in AML cell lines finding that NPM1 partially remained in the nucleolus in the non-A-like NPM1-mutated cells. As a result, only in A-like-mutated cells we detected HOXA5, HOXA10, and HOXB5 hyper-expression and p14ARF/p21/p53 pathway deregulation, leading to reduced sensitivity to the treatment with either chemotherapy or Venetoclax, as compared to non-A-like cells. Overall, we identified that the NPM1 mutational status mediates crucial biological characteristics of AML cells, providing the basis for further sub-classification and, potentially, management of this subgroup of patients.
    Keywords:  HOX genes; Nucleophosmin, NPM1; TP53; acute myeloid leukemia; drug treatment; gene expression; genetic; mutation
  16. Blood. 2021 Jul 21. pii: blood.2020009192. [Epub ahead of print]
      Acute myeloid leukemia (AML) is attractive for the development of CAR T-cell immunotherapy because AML blasts are susceptible to T-cell-mediated elimination. Here, we introduce sialic-acid-binding immunoglobulin-like lectin (Siglec)-6 as a novel target for CAR T-cells in AML. We designed a Siglec-6-specific CAR with a targeting-domain derived from a human monoclonal antibody JML‑1. We found that Siglec-6 is prevalently expressed on AML cell lines and primary AML blasts, including the subpopulation of AML stem cells. Treatment with Siglec-6-CAR T-cells confers specific anti-leukemia reactivity that correlates with Siglec-6-expression in pre-clinical models, including induction of complete remission in a xenograft AML model in immunodeficient mice (NSG/U937). In addition, we confirmed Siglec-6-expression on transformed B-cells in chronic lymphocytic leukemia (CLL) and show specific anti-CLL-reactivity of Siglec-6-CAR T-cells in vitro. Of particular interest, we found that Siglec-6 is not detectable on normal hematopoietic stem and progenitor cells (HSC/P) and that treatment with Siglec-6-CAR T-cells does not affect their viability and lineage differentiation in colony-formation assays. These data suggest that Siglec-6-CAR T-cell therapy may be used to effectively treat AML without a need for subsequent allogeneic hematopoietic stem cell transplantation. In mature normal hematopoietic cells, we detected Siglec-6 in a proportion of memory (and naïve) B-cells and basophilic granulocytes, suggesting the potential for limited on-target/off-tumor reactivity. The lacking expression of Siglec-6 on normal HSC/P is a key differentiator from other Siglec-family members (e.g. Siglec-3=CD33) and other CAR target antigens, e.g. CD123, that are under investigation in AML and warrants the clinical investigation of Siglec-6-CAR T-cell therapy.
  17. Cancers (Basel). 2021 Jul 20. pii: 3629. [Epub ahead of print]13(14):
      P-glycoprotein (known as ABCB1 transporter) expression in myeloid blasts of acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) leads to the commonly observed multidrug resistance. Overexpression of latrophilin-1 was detected in leukemic cells from AML patients. In a previous study, we showed that ABCB1 overexpression is associated with decreased latrophilin-1 expression in MOLM-13/VCR and SKM-1/VCR AML cell variants derived from MOLM-13 and SKM-1 cells by vincristine selection/adaptation. In the present study, we found that if ABCB1 overexpression occurs in myeloid blasts of newly diagnosed MDS patients, latrophilin-1 expression is attenuated. Latrophilin-1 may initiate TIM-3- and galectin-9-mediated immune escape. We demonstrated changes in the expression of both proteins by comparing ABCB1-positive cell variants (MOLM-13/VCR, SKM-1/VCR) with their ABCB1-negative counterparts. Galectin-9 was present in our cell lines in eight protein isoforms for which we identified the respective transcription variants resulting from alternative splicing, and we verified their structure by sequencing. The isoform profile of galectin-9 was different between ABCB1-positive and ABCB1-negative cell variants. The interaction partner of galectin-9 is CD44, and its expression was altered in the ABCB1-positive variants MOLM-13/VCR and SKM-1/VCR compared to their ABCB1-negative counterparts.
    Keywords:  ABCB1; CD44; TIM-3; acute myeloid leukemia; alternative splicing; galectin-9; latrophilin-1; multidrug resistance
  18. Blood Adv. 2021 Jul 27. 5(14): 2901-2908
      Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative therapy for most children with juvenile myelomonocytic leukemia (JMML). Novel therapies controlling the disorder prior to HSCT are needed. We conducted a phase 2, multicenter, open-label study to evaluate the safety and antileukemic activity of azacitidine monotherapy prior to HSCT in newly diagnosed JMML patients. Eighteen patients enrolled from September 2015 to November 2017 were treated with azacitidine (75 mg/m2) administered IV once daily on days 1 to 7 of a 28-day cycle. The primary end point was the number of patients with clinical complete remission (cCR) or clinical partial remission (cPR) after 3 cycles of therapy. Pharmacokinetics, genome-wide DNA-methylation levels, and variant allele frequencies of leukemia-specific index mutations were also analyzed. Sixteen patients completed 3 cycles and 5 patients completed 6 cycles. After 3 cycles, 11 patients (61%) were in cPR and 7 (39%) had progressive disease. Six of 16 patients (38%) who needed platelet transfusions were transfusion-free after 3 cycles. All 7 patients with intermediate- or low-methylation signatures in genome-wide DNA-methylation studies achieved cPR. Seventeen patients received HSCT; 14 (82%) were leukemia-free at a median follow-up of 23.8 months (range, 7.0-39.3 months) after HSCT. Azacitidine was well tolerated and plasma concentration--time profiles were similar to observed profiles in adults. In conclusion, azacitidine monotherapy is a suitable option for children with newly diagnosed JMML. Although long-term safety and efficacy remain to be fully elucidated in this population, these data demonstrate that azacitidine provides valuable clinical benefit to JMML patients prior to HSCT. This trial was registered at as #NCT02447666.
  19. Blood. 2021 Jul 19. pii: blood.2020010477. [Epub ahead of print]
      Antibody-based immunotherapy is a promising strategy for targeting chemo-resistant leukemic cells. However, classical antibody-based approaches are restricted to targeting lineage-specific cell-surface antigens. By targeting intracellular antigens, a large number of other leukemia-associated targets would become accessible. In this study, we evaluated a novel T-cell bispecific (TCB) antibody, generated using CrossMab and knob-into-holes technology, containing a bivalent T-cell receptor-like binding domain that recognizes the RMFPNAPYL peptide derived from the intracellular tumor antigen Wilms' tumor 1 (WT1) in the context of human leukocyte antigen (HLA) A*02. Binding to CD3ε recruits T cells irrespective of their T-cell receptor specificity. WT1-TCB elicited antibody-mediated T-cell cytotoxicity against AML cell lines in a WT1- and HLA-restricted manner. Specific lysis of primary AML cells was mediated in ex vivo long-term co-cultures utilizing allogenic (mean specific lysis: 67±6% after 13-14 days; ±SEM; n=18) or autologous, patient-derived T cells (mean specific lysis: 54±12% after 11-14 days; ±SEM; n=8). WT1-TCB-treated T cells exhibited higher cytotoxicity against primary AML cells than an HLA-A*02 RMF-specific T-cell clone. Combining WT1-TCB with the immunomodulatory drug lenalidomide further enhanced antibody-mediated T-cell cytotoxicity against primary AML cells (mean specific lysis on day 3-4: 45.4±9.0% vs 70.8±8.3%; p=0.015; ±SEM; n=9-10). In vivo, WT1-TCB-treated humanized mice bearing SKM-1 tumors showed a significant and dose-dependent reduction in tumor growth. In summary, we show that WT1-TCB facilitates potent in vitro, ex vivo and in vivo killing of AML cell lines and primary AML cells; these results led to the initiation of a phase I trial in patients with r/r AML (NCT04580121).
  20. Int J Lab Hematol. 2021 Jul;43 Suppl 1 86-95
      CEBPA-mutant acute myeloid leukemia (AML) encompasses clinically and biologically distinct subtypes of AML in both adults and children. CEBPA-mutant AML may occur with monoallelic (moCEBPA) or biallelic (biCEBPA) mutations, which can be somatic or germline, with each entity impacting prognosis in unique ways. BiCEBPA AML is broadly associated with a favorable prognosis, but differences in the type and location of CEBPA mutations as well as the presence of additional leukemogenic mutations can lead to heterogeneity in survival. Concurrent FLT3-ITD mutations have a well-documented negative effect on survival in adult biCEBPA AML, whereas support for a negative prognostic effect of mutations in TET2, DNMT3A, WT1, CSF3R, ASXL1, and KIT is mixed. NPM1 and GATA2 mutations may have a positive prognostic impact. MoCEBPA AML has similar survival outcomes compared to AML with wild-type CEBPA, and risk stratification is determined by other cytogenetic and molecular findings. Germline CEBPA mutations may lead to familial biCEBPA AML after acquisition of second somatic CEBPA mutation, with variable penetrance and age. BiCEBPA AML in children is likely a favorable-risk diagnosis as it is in adults, but the role of a single CEBPA mutation and the impact of concurrent leukemogenic mutations are not clear in this population. Laboratory evaluation of the CEBPA gene includes PCR-based fragment-length analysis, Sanger sequencing, and next-generation sequencing. Phenotypic analysis using multiparameter flow cytometry can also provide additional data in evaluating CEBPA, helping to assess for the likelihood of mutation presence.
    Keywords:  AML; CEBPA; flow cytometry; molecular diagnosis; sequencing
  21. J Cancer Res Clin Oncol. 2021 Jul 23.
      BACKGROUND: Acute myeloid leukemia (AML) with antecedent hematological disease (s-AML) and treatment-related AML (t-AML) predicts poor prognosis. Intensive treatment protocols of those high-risk patients should consider allogeneic stem cell transplantation (allo-HSCT) in first complete remission (CR). Despite allo-HSCT, relapse rate remains high. Induction chemotherapy with liposomal cytarabine and daunorubicin (CPX-351) has been approved for patients with AML with myeloid-related changes (AML-MRC) or t-AML based on improved survival and remission rates compared to standard 7 + 3 induction.PATIENTS AND METHODS: 110 patients with newly diagnosed s-AML or t-AML at a university hospital were analyzed retrospectively. Median age was 62 years (24-77 years). A total of 65 patients with s-AML after MDS (59%) and 23 patients (20.9%) with t-AML were included. Induction chemotherapy consisted of intermediate-dosed cytarabine (ID-AraC) in combination with idarubicin (patients up to 60 years) or mitoxantrone (patients over 60 years). In patients subsequently undergoing allo-HSCT, reduced conditioning regimens (RIC) were applied prior to transplantation in 47 of 62 patients (76%).
    RESULTS: Induction chemotherapy with ID-AraC resulted in an overall response rate of 83% including complete remission (CR/CRi) in 69 patients (63%) with a low rate of early death (2.7%). Most relevant non-hematologic toxicity consisted of infectious complications including sepsis with need of intensive care treatment in five patients (4.5%) and proven or probable invasive fungal disease in eight patients (7.2%). Relapse-free survival (RFS), event-free survival (EFS) and overall survival (OS) of the whole cohort were 19 months (0-167), 10 months (0-234) and 15 months (0-234), respectively (p < 0.0001). A significant improvement of OS was observed in patients who underwent allo-HSCT compared to those without subsequent allo-HSCT: 9 vs. 46 months, p < 0.0001. Rate of transplantation-related mortality (TRM) in the early phase post allo-HSCT was low (0.9% at day 30 and 1.8% at day 90, respectively). RIC conditioning results in OS rate of 60% after 60 months post allo-HSCT (median OS not reached).
    CONCLUSION: S-AML and t-AML patients receiving induction chemotherapy with intermediate-dosed cytarabine showed satisfactory response rate and consolidation therapy with allo-HSCT after full or reduced-intensity conditioning further improved survival in these patients with similar outcome as reported for CPX-351.
    Keywords:  Allo-HSCT; CPX-351; High-risk AML; Induction chemotherapy; MDS; RIC
  22. Br J Haematol. 2021 Jul 22.
      In myelodysplastic syndromes (MDS), the 20q deletion [del(20q)] may cause deletion of the ASXL1 gene. We studied 153 patients with MDS and del(20q) to assess the incidence, prognostic value and impact on response to azacitidine (AZA) of ASXL1 chromosomal alterations and genetic mutations. Additionally, in vitro assay of the response to AZA in HAP1 (HAP1WT ) and HAP1 ASXL1 knockout (HAP1KN ) cells was performed. ASXL1 chromosomal alterations were detected in 44 patients (28·5%): 34 patients (22%) with a gene deletion (ASXL1DEL ) and 10 patients (6·5%) with additional gene copies. ASXL1DEL was associated with a lower platelet count. The most frequently mutated genes were U2AF1 (16%), ASXL1 (14%), SF3B1 (11%), TP53 (7%) and SRSF2 (6%). ASXL1 alteration due to chromosomal deletion or genetic mutation (ASXL1DEL /ASXL1MUT ) was linked by multivariable analysis with shorter overall survival [hazard ratio, (HR) 1·84; 95% confidence interval, (CI): 1·11-3·04; P = 0·018] and a higher rate for acute myeloid leukaemia progression (HR 2·47; 95% CI: 1·07-5·70, P = 0·034). ASXL1DEL /ASXL1MUT patients were correlated by univariable analysis with a worse response to AZA. HAP1KN cells showed more resistance to AZA compared to HAP1WT cells. In conclusion, ASXL1 alteration exerts a negative impact on MDS with del(20q) and could become useful for prognostic risk stratification and treatment decisions.
    Keywords:   ASXL1 ; 20q deletion; azacitidine; gene mutations; myelodysplastic syndromes
  23. Cancers (Basel). 2021 Jul 06. pii: 3385. [Epub ahead of print]13(14):
      Despite progress in the treatment of acute myeloid leukemia (AML), the clinical outcome remains suboptimal and many patients are still dying from this disease. First-line treatment consists of chemotherapy, which typically includes cytarabine (AraC), either alone or in combination with anthracyclines, but drug resistance can develop and significantly worsen prognosis. Better treatments are needed. We are developing a novel anticancer compound, NEO212, that was created by covalent conjugation of two different molecules with already established anticancer activity, the alkylating agent temozolomide (TMZ) and the natural monoterpene perillyl alcohol (POH). We investigated the anticancer activity of NEO212 in several in vitro and in vivo models of AML. Human HL60 and U937 AML cell lines, as well as different AraC-resistant AML cell lines, were treated with NEO212 and effects on cell proliferation, cell cycle, and cell death were investigated. Mice with implanted AraC-sensitive or AraC-resistant AML cells were dosed with oral NEO212, and animal survival was monitored. Our in vitro experiments show that treatment of cells with NEO212 results in growth inhibition via potent G2 arrest, which is followed by apoptotic cell death. Intriguingly, NEO212 was equally potent in highly AraC-resistant cells. In vivo, NEO212 treatment strikingly extended survival of AML mice and the majority of treated mice continued to thrive and survive without any signs of illness. At the same time, we were unable to detect toxic side effects of NEO212 treatment. All in all, the absence of side effects, combined with striking therapeutic activity even in an AraC-resistant context, suggests that NEO212 should be developed further toward clinical testing.
    Keywords:  DNA alkylation; acute myeloid leukemia; cytarabine resistance; perillyl alcohol
  24. Eur J Haematol. 2021 Jul 20.
      OBJECTIVES: Maintenance therapy is one strategy to prolong survival in patients with acute myeloid leukemia (AML) following hematopoietic stem cell transplantation (HSCT). We evaluated real-world treatment patterns and outcomes in patients with newly diagnosed FLT3-mutated AML receiving HSCT after complete remission with first-line chemotherapy.METHODS: A global, retrospective chart review to evaluate maintenance therapy and outcomes in patients with FLT3-mutated AML after HSCT.
    RESULTS: Data from 1,208 charts from eight countries showed that most patients (n=765 [63.3%]) received no maintenance therapy after HSCT, 219 (18.1%) received FLT3 inhibitor maintenance therapy, and 224 (18.5%) received other types of maintenance therapy. No systematic differences were observed in healthcare resource utilization across the three groups. Clinical benefit was observed with FLT3 inhibitor maintenance over no maintenance therapy with relapse-free survival (adjusted hazard ratio [HR] 0.57 [95% CI 0.34-0.94], P<0.05). FLT3 inhibitor and other maintenance also demonstrated overall survival benefit over no maintenance (adjusted HR 0.50 [95% CI 0.28-0.89] and 0.46 [95% CI 0.23-0.91], respectively; both P<0.05).
    CONCLUSIONS: Real-world maintenance therapies after HSCT in patients with FLT3-mutated AML were heterogeneous. While overall use of healthcare resources was not significantly increased in patients receiving maintenance therapy versus those who did not, clinical outcomes were improved.
    Keywords:  Acute; Health Care; Hematopoietic Stem Cell Transplantation; Leukemia; Myeloid; Outcome Assessment
  25. Leuk Lymphoma. 2021 Jul 22. 1-11
      Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection produces higher morbidity and mortality in hematological malignancies, but evidence in acute myeloid leukemia (AML) is scarce. A multicenter observational study was conducted to determine the clinical outcomes and assess the impact of therapeutic approaches in adult AML patients with SARS-CoV-2 infection in the first wave (March-May 2020). Overall, 108 patients were included: 51.9% with active leukemia and 70.4% under therapeutic schedules for AML. Signs and symptoms of SARS-CoV-2 were present in 96.3% of patients and 82.4% received specific treatment for SARS-CoV-2. The mortality rate was 43.5% and was correlated with age, gender, active leukemia, dyspnea, severe SARS-CoV-2, intensive care measures, neutrophil count, and D-dimer levels. A protective effect was found with azithromycin, lopinavir/ritonavir, and normal liver enzyme levels. During the SARS-CoV-2 first wave, our findings suggested an increased mortality in AML in a short period. SARS-CoV-2 management could be guided by risk factors in AML patients.
    Keywords:  COVID-19; SARS-CoV-2; acute myeloid leukemia; hematological malignancies
  26. Stem Cell Res. 2021 Jul 12. pii: S1873-5061(21)00311-1. [Epub ahead of print]55 102464
      Chronic myeloid leukemia (CML) is caused by the BCR-ABL fusion protein, which dysregulates tyrosine kinase activity. In this study, we generated induced pluripotent stem cells (iPSCs) carrying the Philadelphia chromosome from a CML patient with the BCR-ABL fusion protein. CML iPSCs were positive for pluripotency markers and had the ability to differentiate into the three germ layers. This iPSC cell line could be useful for studying CML pathogenesis as well as for drug development to treat CML.
    Keywords:  Chronic myeloid leukemia; Induced pluripotent stem cells; Philadelphia chromosome
  27. Ann Hematol. 2021 Jul 19.
      Recent studies have shown that approximately 50% of patients with chronic myeloid leukemia (CML) receiving tyrosine kinase inhibitor (TKI) therapy with a sustained deep molecular response (DMR) (BCR-ABL1IS ≤ 0.01%) can achieve treatment-free remission (TFR, stopping TKI without relapse) and that prior interferon (IFN)-α therapy and higher NK cell counts at and after TKI discontinuation are associated with TFR. We recently reported that post-TKI discontinuation of IFN-α therapy could prevent molecular relapse (MR, BCR-ABL1IS > 0.1%). Here, we evaluated whether NK cells are associated with MR and investigated the effects of post-TKI discontinuation IFN-α therapy on lymphocyte subsets. A total of 34 patients measuring blood lymphocyte subclasses were included. In the 22 patients who did not receive IFN-α therapy, at 1 month after TKI discontinuation, the nonrelapsed patients showed a significantly higher proportion and count of NK cells than the relapsed patients. In particular, the proportion and count of CD56dim NK cells were significantly higher in the nonrelapsed patients than in the relapsed patients. In the 12 patients who received IFN-α therapy, the level of CD56bright NK cells increased significantly after 3 and 6 months of IFN-α therapy. In summary, NK cells, in particular CD56dim NK cells, were associated with MR after TKI discontinuation in patients with CML. Additionally, IFN-α therapy gradually increased the level of CD56bright NK cells in patients with CML.
    Keywords:  Chronic myeloid leukemia; Interferon; Natural killer cell; Treatment-free remission; Tyrosine kinase inhibitor
  28. Bone Marrow Transplant. 2021 Jul 17.
      We retrospectively compared the outcomes of 20 patients receiving Venetoclax + low-dose Cytarabine + Actinomycin D (ACTIVE) with 29 patients receiving FLAG-Ida as salvage therapy for relapsed or refractory AML (R/R AML) after alloSCT. The groups were statistically balanced according to age, performance status, cytogenetics, and previous treatment. The overall response rate (CR + CRp + MLFS) of ACTIVE was 75% (15/20) in comparison to 66% (19/29) in the FLAG-Ida group (p = 0.542). The cumulative CR + CRp rate was significantly higher in the ACTIVE group compared to FLAG-Ida (70% (14/20) vs. 34% (10/29), respectively, p = 0.02). All three patients failing previous Venetoclax therapy and five out of seven patients with previous FLAG-Ida exposure achieved a CR/CRp after ACTIVE induction. ACTIVE patients survived longer compared to FLAG-Ida patients (13.1 vs. 5.1 months, respectively, p = 0.032). The treatment-related mortality was 0% in the ACTIVE group and 34% (10/29) in the FLAG-Ida patients (p = 0.003). The cumulative incidence of relapse did not differ between the two treatment groups. ACTIVE appears to have comparable antileukemic activity and lower toxicity compared to FLAG-Ida resulting in improved survival. Patients with Venetoclax or FLAG-Ida exposure responded to ACTIVE.
  29. J Clin Oncol. 2021 Jul 20. JCO2002724
      PURPOSE: In contrast to 5-azacytidine (5-aza), allogeneic stem-cell transplantation (HSCT) represents a curative treatment strategy for patients with myelodysplastic syndromes (MDS), but therapy-related mortality (TRM) limits its broader use in elderly patients with MDS. The present prospective multicenter study compared HSCT following 5-aza pretreatment with continuous 5-aza treatment in patients with higher-risk MDS age 55-70 years.METHODS: One hundred ninety patients with a median age of 63 years were enrolled. Patients received 4-6 cycles of 5-aza followed by HLA-compatible HSCT after reduced-intensity conditioning or by continuous 5-aza if no donor was identified.
    RESULTS: Twenty-eight patients did not fulfill inclusion criteria (n = 20), died (n = 2) withdrew informed consent (n = 5), or were excluded for an unknown reason (n = 1). 5-aza induction started in 162 patients, but only 108 (67%) were eligible for subsequent allocation to HSCT (n = 81) or continuation of 5-aza (n = 27) because of disease progression (n = 26), death (n = 12), or other reasons (n = 16). Seven percent died during 5-aza before treatment allocation. The cumulative incidence of TRM after HSCT at 1 year was 19%. The event-free survival and overall survival after 5-aza pretreatment and treatment allocation at 3 years were 34% (95% CI, 22 to 47) and 50% (95% CI, 39 to 61) after allograft and 0% and 32% (95% CI, 14 to 52) after continuous 5-aza treatment (P < .0001 and P = .12), respectively. Fourteen patients progressing after continuous 5-aza received a salvage allograft from an alternative donor, and 43% were alive at last follow-up.
    CONCLUSION: In older patients with MDS, reduced-intensity conditioning HSCT resulted in a significantly improved event-free survival in comparison with continuous 5-aza therapy. Bridging with 5-aza to HSCT before is associated with a considerable rate of dropouts because of progression, mortality, and adverse events.
  30. Leuk Lymphoma. 2021 Jul 21. 1-11
      Relapse is the most common cause of treatment failure following allogeneic blood or marrow transplantation (alloBMT) for AML or MDS. Post-transplant maintenance therapies may prevent relapse. We conducted a phase II trial combining azacitidine (AZA) with GM-CSF in non-relapsed, post-transplant patients with AML or MDS. Patients received escalating doses of AZA to a maximum of 75 mg/m2 for 5 days per cycle for up to 12 cycles. GM-CSF was given on days 1-10 of each cycle. Eighteen patients were treated following non-myeloablative (17) and myeloablative (1) alloBMT for AML (61.1%), MDS (27.7%), or therapy-related myeloid neoplasm (11.1%). The majority of patients (72%) received their graft from an HLA-haploidentical donor. The treatment was well-tolerated with rare grade 3-4 hematologic toxicities. One patient suffered an exacerbation of GVHD. The 24-month relapse-free and overall survivals were 47 and 57%, respectively, with a median of 18.6 and 29 months.
    Keywords:  AML; Azacitidine; GM-CSF; MDS; Post-transplant maintenance
  31. Blood. 2021 Jul 19. pii: blood.2021011787. [Epub ahead of print]
      Asparaginase (ASNase) therapy has been a mainstay of Acute Lymphoblastic Leukemia (ALL) protocols for decades and shows promise in the treatment of a variety of other cancers. To improve the efficacy of ASNase treatment, we employed a CRISPR/Cas9-based screen to identify actionable signaling intermediates that improve the response to ASNase. Both genetic inactivation of Bruton's Tyrosine Kinase (BTK) and pharmacological inhibition by the BTK inhibitor ibrutinib strongly synergize with ASNase by inhibiting the amino acid response pathway, a mechanism involving c-Myc mediated suppression of GCN2 activity. This synthetic lethal interaction was observed in 90% of patient derived xenografts, irrespective of the genomic subtype. Moreover, ibrutinib substantially improved ASNase treatment response in a murine PDX model. Hence, ibrutinib may be used to enhance the clinical efficacy of ASNase in ALL.