bims-tremyl Biomed News
on Therapy resistance biology in myeloid leukemia
Issue of 2020‒10‒04
nineteen papers selected by
Paolo Gallipoli
Barts Cancer Institute, Queen Mary University of London

  1. Biomedicines. 2020 Sep 28. pii: E385. [Epub ahead of print]8(10):
      All-trans retinoic acid (atRA) has a dramatic impact on the survival of patients with acute promyelocytic leukemia, but its therapeutic value in other types of acute myeloid leukemia (AML) has so far remained unclear. Given that AML is a stem cell-driven disease, recent studies have addressed the effects of atRA on leukemic stem cells (LSCs). atRA promoted stemness of MLL-AF9-driven AML in an Evi1-dependent manner but had the opposite effect in Flt3-ITD/Nup98-Hoxd13-driven AML. Overexpression of the stem cell-associated transcription factor EVI1 predicts a poor prognosis in AML, and is observed in different genetic subtypes, including cytogenetically normal AML. Here, we therefore investigated the effects of Evi1 in a mouse model for cytogenetically normal AML, which rests on the combined activity of Flt3-ITD and Npm1c mutations. Experimental expression of Evi1 on this background strongly promoted disease aggressiveness. atRA inhibited leukemia cell viability and stem cell-related properties, and these effects were counteracted by overexpression of Evi1. These data further underscore the complexity of the responsiveness of AML LSCs to atRA and point out the need for additional investigations which may lay a foundation for a precision medicine-based use of retinoids in AML.
    Keywords:  AML; EVI1; FLT3-ITD; MECOM; all-trans retinoic acid; leukemia stem cells
  2. Blood Adv. 2020 Oct 13. 4(19): 4623-4631
      Mixed-lineage leukemia (MLL) gene rearrangements are among the most frequent chromosomal abnormalities in acute myeloid leukemia (AML). MLL fusion patterns are associated with the patient's prognosis; however, their relationship with driver mutations is unclear. We conducted sequence analyses of 338 genes in pediatric patients with MLL-rearranged (MLL-r) AML (n = 56; JPLSG AML-05 study) alongside data from the TARGET study's pediatric cohorts with MLL-r AML (n = 104), non-MLL-r AML (n = 581), and adult MLL-r AML (n = 81). KRAS mutations were most frequent in pediatric patients with high-risk MLL fusions (MLL-MLLLT10, MLL-MLLT4, and MLL-MLLT1). Pediatric patients with MLL-r AML (n = 160) and a KRAS mutation (KRAS-MT) had a significantly worse prognosis than those without a KRAS mutation (KRAS-WT) (5-year event-free survival [EFS]: 51.8% vs 18.3%, P < .0001; 5-year overall survival [OS]: 67.3% vs 44.3%, P = .003). The adverse prognostic impact of KRAS mutations was confirmed in adult MLL-r AML. KRAS mutations were associated with adverse prognoses in pediatric patients with both high-risk (MLLT10+MLLT4+MLLT1; n = 60) and intermediate-to-low-risk (MLLT3+ELL+others; n = 100) MLL fusions. The prognosis did not differ significantly between patients with non-MLL-r AML with KRAS-WT or KRAS-MT. Multivariate analysis showed the presence of a KRAS mutation to be an independent prognostic factor for EFS (hazard ratio [HR], 2.21; 95% confidence interval [CI], 1.35-3.59; P = .002) and OS (HR, 1.85; 95% CI, 1.01-3.31; P = .045) in MLL-r AML. The mutation is a distinct adverse prognostic factor in MLL-r AML, regardless of risk subgroup, and is potentially useful for accurate treatment stratification. This trial was registered at the UMIN (University Hospital Medical Information Network) Clinical Trials Registry (UMIN-CTR; as #UMIN000000511.
  3. Onco Targets Ther. 2020 ;13 7887-7896
      Background: Acute myeloid leukemia (AML) is a group of malignant hematopoietic system diseases. Taurine-upregulated gene 1 (TUG1) is a long non-coding RNA that has been associated with human cancers, including AML. However, the role and molecular mechanisms of TUG1 in AML remains to be defined.Methods: Expression of TUG1 and miR-185 was detected using RT-qPCR. Cell viability and apoptotic rate were measured by MTT assay and flow cytometry, respectively. Glycolysis was determined by commercial glucose and lactate assay kits and Western blot. The target binding between TUG1 and miR-185 was predicted on Starbase online database and confirmed by luciferase reporter assay and RNA immunoprecipitation.
    Results: TUG1 was upregulated and miR-185 was downregulated in the peripheral blood mononuclear cells of AML specimens and cells (HL-60, KG-1, MOLM-14, and MOLM-13). Both TUG1 knockdown and miR-185 overexpression via transfection could suppress cell viability, glucose consumption, lactate production, and hexokinase 2 expression, but promote apoptotic rate in HL-60 and KG-1 cells. Notably, TUG1 functioned as a sponge of miR-185 by target binding. Moreover, downregulation of miR-185 could partially overturn the effect of TUG1 knockdown on cell proliferation and glycolysis in HL-60 and KG-1 cells.
    Conclusion: Expression of TUG1 was upregulated in AML patients and cells, and its knockdown repressed cell proliferation and glycolysis in AML cells in vitro by targeting miR-185.
    Keywords:  AML; TUG1; glycolysis; miR-185
  4. Cancer Immunol Immunother. 2020 Sep 29.
      CD47 is over-expressed in Acute Myeloid Leukemia (AML) and functions as an inhibitory signal, suppressing phagocytosis by binding to signal regulatory protein α (SIRPα) on the surface of macrophages. Inhibition of CD47 restores the immune surveillance of AML cells. However, the inhibition of CD47 in AML by activated macrophages and the subsequent effects on different immune response parameters are not fully understood. Here, we demonstrate the use of a distinct co-culture method to inhibit CD47 and therefore eliminate AML cells by macrophages in vitro. Human chemically induced THP-1 macrophages were activated using different concentrations of lipopolysaccharide (LPS) and co-culturing with three AML cancer cell lines (HL-60, NB4, and THP-1), respectively, as well as normal human peripheral blood mononuclear cells (PBMC). CD47 inhibition was observed in and selective to AML but not observed in normal PBMC. Additionally, calreticulin (CRT) levels were elevated in the same cell lines simultaneously, after co-culturing with activated human macrophages, but not elevated in normal cells. We also show that the activated macrophages secreted high levels of cytokines, including IL-12p70, IL-6, and TNF-α, consistent with the elimination of AML by macrophages. Our study reveals the potential of this model for screening new drugs against AML and the possibility of using human macrophages in AML treatment in the future.
    Keywords:  Acute myeloid leukemia (AML); CD47; Calreticulin; Co-culture; Lipopolysaccharide; Stimulated-macrophage phagocytosis
  5. Cancers (Basel). 2020 Sep 29. pii: E2806. [Epub ahead of print]12(10):
      Fms-like tyrosine kinase 3 (FLT3) is a member of the class III receptor tyrosine kinases (RTK) and is involved in cell survival, proliferation, and differentiation of haematopoietic progenitors of lymphoid and myeloid lineages. Oncogenic mutations in the FLT3 gene resulting in constitutively active FLT3 variants are frequently found in acute myeloid leukaemia (AML) patients and correlate with patient's poor survival. Targeting FLT3 mutant leukaemic stem cells (LSC) is a key to efficient treatment of patients with relapsed/refractory AML. It is therefore essential to understand how LSC escape current therapies in order to develop novel therapeutic strategies. Here, we summarize the current knowledge on mechanisms of FLT3 activity regulation and its cellular consequences. Furthermore, we discuss how aberrant FLT3 signalling cooperates with other oncogenic lesions and the microenvironment to drive haematopoietic malignancies and how this can be harnessed for therapeutical purposes.
    Keywords:  FMS-like tyrosine kinase 3 (FLT3); acute myeloid leukaemia (AML); cancer cell vulnerability; haematopoietic niche; oncogenic signaling; re-sistance development
  6. Cancers (Basel). 2020 Sep 26. pii: E2766. [Epub ahead of print]12(10):
      NUP98-NSD1-positive acute myeloid leukemia (AML) is a poor prognostic subgroup that is frequently diagnosed in pediatric cytogenetically normal AML. NUP98-NSD1-positive AML often carries additional mutations in genes including FLT3, NRAS, WT1, and MYC. The purpose of our study was to characterize the cooperative potential of the fusion and its associated Neuroblastoma rat sarcoma (NRAS) mutation. By constitutively expressing NUP98-NSD1 and NRASG12D in a syngeneic mouse model and using a patient-derived xenograft (PDX) model from a NUP98-NSD1-positive AML patient, we evaluated the functional role of these genes and tested a novel siRNA formulation that inhibits the oncogenic driver NUP98-NSD1. NUP98-NSD1 transformed murine bone marrow (BM) cells in vitro and induced AML in vivo. While NRASG12D expression was insufficient to transform cells alone, co-expression of NUP98-NSD1 and NRASG12D enhanced the leukemogenicity of NUP98-NSD1. We developed a NUP98-NSD1-targeting siRNA/lipid nanoparticle formulation that significantly prolonged the survival of the PDX mice. Our study demonstrates that mutated NRAS cooperates with NUP98-NSD1 and shows that direct targeting of the fusion can be exploited as a novel treatment strategy in NUP98-NSD1-positive AML patients.
    Keywords:  AML; NRASG12D; NUP98-NSD1; liposome; siRNA
  7. Leuk Lymphoma. 2020 Sep 30. 1-11
      Chromosomal translocations and rearrangements involving Mixed Lineage Leukemia (MLL) gene is associated with poor prognosis in AML. Extensive epigenetic changes were found in this group of patients. In clinical study, we found miR-181a expression level was significantly lower in MLL-rearranged AML. As an important epi-miRNA, the role of miR-181a as an epigenetic regulator in leukemia has not been investigated before. In this study, we found miR-181a overexpression enhanced total protein acetylation in THP-1 cells, which harbor MLL-AF9 fusion gene, and protein Mass Spectrum identified poly(ADP-ribose) polymerase 1 (PARP1) was a major downstream target. Increased PARP1 acetylation was mediated by down-regulation of histone deacetylase Sirtuin1 (Sirt1). MiR-181a overexpression resulted in DNA trapping of PARP1, increased DNA double strand break formation and increased chemosensitivity of leukemia cells both in vitro and in vivo. This study indicates miR-181a-Sirt1-PARP1 acetylation pathway could be a promising target for this special group of AML.
    Keywords:  AML; PARP1; Sirt1; acetylation; miR-181a
  8. Clin Cancer Res. 2020 Sep 30. pii: clincanres.2586.2020. [Epub ahead of print]
      PURPOSE: The Polo-like kinase 1 (PLK1) is a master regulator of mitosis and overexpressed in acute myeloid leukemia (AML). We conducted a phase 1b study of the PLK1 inhibitor onvansertib in combination with either low-dose cytarabine (LDAC) or decitabine in relapsed or refractory (R/R) AML patients.EXPERIMENTAL DESIGN: Onvansertib was administered orally, in escalating doses, on days 1-5 in combination with either LDAC (20 mg/m2; days 1-10) or decitabine (20 mg/m2; days 1-5) in a 28-day cycle. The primary endpoint was to evaluate first-cycle dose-limiting toxicities and the maximum tolerated dose (MTD). Secondary and exploratory endpoints included safety, pharmacokinetics, anti-leukemic activity and response biomarkers.
    RESULTS: Forty patients were treated with onvansertib (12-90 mg/m2) in combination with LDAC (n=17) or decitabine (n=23). Onvansertib was well tolerated with most grades 3 and 4 adverse events related to myelosuppression. In the decitabine arm, the MTD was established at 60 mg/m2, and 5 (24%) of the 21 evaluable patients achieved complete remission with or without hematologic count recovery. Decrease in mutant circulating tumor DNA (ctDNA) during the first cycle of therapy was associated with clinical response. Engagement of the PLK1 target, TCTP, was measured in circulating blasts and was associated with greater decrease in bone marrow blasts.
    CONCLUSIONS: The onvansertib and decitabine combination was well tolerated and had anti-leukemic activity particularly in patients with target engagement and decreased mutant ctDNA following treatment. This combination will be further investigated in the ongoing phase 2 trial.
  9. BMC Pharmacol Toxicol. 2020 Sep 29. 21(1): 70
      BACKGROUND: Combined inhibition of phosphatidylinositol 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) complexes may be an efficient treatment for acute leukemia. The primary objective of this phase I single center open label study was to determine the maximum tolerated dose (MTD) and recommended phase II dose (RP2D) of the dual pan-class I PI3K and mTOR inhibitor BEZ235 in patients with advanced leukemia.METHODS: Herein patients > 18 years of age who had relapsed or showed refractory leukemia were treated with BEZ235 (orally at 300-400 mg BID (cohort - 1/1)) to assess safety, tolerability, preliminary efficacy and pharmacokinetic (PK). Adverse events data and serious adverse events were analyzed and haematological and clinical biochemistry toxicities were assessed from laboratory test parameters. Response was assessed for the first time at the end of cycle 1 (day 29) and after every subsequent cycle. Pharmacokinetic and pharmacodynamic analyses of BEZ235 were also included (BEZ235 plasma levels, phosphorylation of AKT, S6 and 4EBP1). On statistics this trial is a multiple ascending dose study in which a following variant of the 3 + 3 rule ("Rolling Six"), a minimum of 6 and a maximum of 12 patients was recruited for the dose escalation and another 5 were planned for the expansion phase.
    RESULTS: Twenty-four patients with ALL (n = 11) or AML (n = 12) or CML-BP (n = 1) were enrolled. All patients had failed one (n = 5) or more lines of therapy (n = 5) and 14 patients were in refractory / refractory relapse. No formal MTD was defined, stomatitis and gastrointestinal toxicity at 400 mg BID dose was considered incompatible with prolonged treatment. The RP2D of BEZ235 was defined as 300 mg BID. Four of 24 patients showed clinical benefit. Twenty-two of 24 patients discontinued because of progression, (median time to progression 27 days (4d-112d). There was no association between PK parameters and efficacy or tolerability.
    CONCLUSIONS: Combined inhibition of PI3K and mTOR inhibits a clinically meaningful driver pathway in a small subset of patients with ALL, with no benefit in patients with AML.
    TRIAL REGISTRATION: , identifier NCT01756118. retrospectively registered 19th December 2012, .
    Keywords:  BEZ235; PI3K/mTor inhibition; Phase I clinical trial; Refractory ALL; Refractory AML
  10. STAR Protoc. 2020 Sep 18. pii: 100097. [Epub ahead of print]1(2):
      A combination of immunotherapy and chemotherapy strategies could strengthen antitumor effects. This protocol elucidates a robust method via co-culturing drug pre-treated acute myeloid leukemia cells with CD3+ T cells, derived from leukoreduction system chambers, for in vitro and in vivo study. We optimized several aspects of the procedures, including timing of drug treatment, quantification of tumor cells, and approach of combination of CD3+ T cells with drug treatment in vivo. This enables the readouts of the interplay between drugs and T cells. For complete details on the use and execution of this protocol, please refer to Su et al. (2020).
  11. Genes Dev. 2020 Oct 01.
      EBF1 and PAX5 mutations are associated with the development of B progenitor acute lymphoblastic leukemia (B-ALL) in humans. To understand the molecular networks driving leukemia in the Ebf1+/-Pax5+/- (dHet) mouse model for B-ALL, we interrogated the transcriptional profiles and chromatin status of leukemic cells, preleukemic dHet pro-B, and wild-type pro-B cells with the corresponding EBF1 and Pax5 cistromes. In dHet B-ALL cells, many EBF1 and Pax5 target genes encoding pre-BCR signaling components and transcription factors were down-regulated, whereas Myc and genes downstream from IL-7 signaling or associated with the folate pathway were up-regulated. We show that blockade of IL-7 signaling in vivo and methotrexate treatment of leukemic cells in vitro attenuate the expansion of leukemic cells. Single-cell RNA-sequencing revealed heterogeneity of leukemic cells and identified a subset of wild-type pro-B cells with reduced Ebf1 and enhanced Myc expression that show hallmarks of dHet B-ALL cells. Thus, EBF1 and Pax5 may safeguard early stage B cells from transformation to B-ALL by limiting IL-7 signaling, folate metabolism and Myc expression.
    Keywords:  B-ALL; EBF1; IL-7 signaling; Pax5; cMyc; folate pathway
  12. PLoS One. 2020 ;15(10): e0238795
      Hypomethylating agents are a classical frontline low-intensity therapy for older patients with acute myeloid leukemia. Recently, TP53 gene mutations have been described as a potential predictive biomarker of better outcome in patients treated with a ten-day decitabine regimen., However, functional characteristics of TP53 mutant are heterogeneous, as reflected in multiple functional TP53 classifications and their impact in patients treated with azacitidine is less clear. We analyzed the therapeutic course and outcome of 279 patients treated with azacitidine between 2007 and 2016, prospectively enrolled in our regional healthcare network. By screening 224 of them, we detected TP53 mutations in 55 patients (24.6%), including 53 patients (96.4%) harboring high-risk cytogenetics. The identification of any TP53 mutation was associated with worse overall survival but not with response to azacitidine in the whole cohort and in the subgroup of patients with adverse karyotype. Stratification of patients according to three recent validated functional classifications did not allow the identification of TP53 mutated patients who could benefit from azacitidine. Systematic TP53 mutant classification will deserve further exploration in the setting of patients treated with conventional therapy and in the emerging field of therapies targeting TP53 pathway.
  13. Cell Rep. 2020 Sep 29. pii: S2211-1247(20)31189-X. [Epub ahead of print]32(13): 108200
      Uncontrolled self-renewal of hematopoietic progenitors induces leukemia. To self-renew, leukemia cells must continuously activate genes that were previously active in their mother cells. Here, we describe the circuitry of a transactivation system responsible for oncogenic self-renewal. MLL recruits RNA polymerase II (RNAP2) to unmethylated CpG-rich promoters by its CXXC domain and activates transcription by transcriptional regulators, including the AF4 family/ENL family/P-TEFb complex, DOT1L, and p300/CBP histone acetyl transferases. MOZ also targets a broad range of CpG-rich promoters through association with RNAP2 and MLL. Leukemic fusion proteins such as MOZ-TIF2 and MLL-AFX constitutively activate CpG-rich promoters by aberrantly recruiting p300/CBP. Pharmacological inhibition of MLL or DOT1L induces differentiation of MOZ-TIF2-transformed cells. These results reveal that activation of unmethylated CpG-rich promoters mediated by MLL is the central mechanism of oncogenic self-renewal in MOZ-rearranged leukemia and indicate that the molecularly targeted therapies intended for MLL-rearranged leukemia can be applied for MOZ-rearranged leukemia.
    Keywords:  CXXC domain; ENL; MLL; MOZ; RNA polymerase II; histone acetyl transferase; leukemia; self-renewal; transcription; unmethylated CpG
  14. Nat Commun. 2020 09 28. 11(1): 4886
      Somatic mutations in the calreticulin (CALR) gene are associated with approximately 30% of essential thrombocythemia (ET) and primary myelofibrosis (PMF). CALR mutations, including the two most frequent 52 bp deletion (del52) and 5 bp insertion (ins5), induce a frameshift to the same alternative reading frame generating new C-terminal tails. In patients, del52 and ins5 induce two phenotypically distinct myeloproliferative neoplasms (MPNs). They are equally found in ET, but del52 is more frequent in PMF. We generated heterozygous and homozygous conditional inducible knock-in (KI) mice expressing a chimeric murine CALR del52 or ins5 with the human mutated C-terminal tail to investigate their pathogenic effects on hematopoiesis. Del52 induces greater phenotypic changes than ins5 including thrombocytosis, leukocytosis, splenomegaly, bone marrow hypocellularity, megakaryocytic lineage amplification, expansion and competitive advantage of the hematopoietic stem cell compartment. Homozygosity amplifies these features, suggesting a distinct contribution of homozygous clones to human MPNs. Moreover, homozygous del52 KI mice display features of a penetrant myelofibrosis-like disorder with extramedullary hematopoiesis linked to splenomegaly, megakaryocyte hyperplasia and the presence of reticulin fibers. Overall, modeling del52 and ins5 mutations in mice successfully recapitulates the differences in phenotypes observed in patients.
  15. Cancer Discov. 2020 Oct;10(10): 1445-1447
      In this issue of Cancer Discovery, Cai and colleagues delineate a new mechanism that links cell of origin, the transcription factor EVI1, apoptotic priming, and therapeutic susceptibility in mixed lineage leukemia-rearranged acute myeloid leukemia. These findings establish a cell of origin-dependent program that may be leveraged by therapeutic combinations to overcome drug resistance in chemoresistant leukemias.See related article by Cai et al., p. 1500.
  16. Nat Chem Biol. 2020 Sep 28.
      The mitotic kinase AURORA-A is essential for cell cycle progression and is considered a priority cancer target. Although the catalytic activity of AURORA-A is essential for its mitotic function, recent reports indicate an additional non-catalytic function, which is difficult to target by conventional small molecules. We therefore developed a series of chemical degraders (PROTACs) by connecting a clinical kinase inhibitor of AURORA-A to E3 ligase-binding molecules (for example, thalidomide). One degrader induced rapid, durable and highly specific degradation of AURORA-A. In addition, we found that the degrader complex was stabilized by cooperative binding between AURORA-A and CEREBLON. Degrader-mediated AURORA-A depletion caused an S-phase defect, which is not the cell cycle effect observed upon kinase inhibition, supporting an important non-catalytic function of AURORA-A during DNA replication. AURORA-A degradation induced rampant apoptosis in cancer cell lines and thus represents a versatile starting point for developing new therapeutics to counter AURORA-A function in cancer.
  17. Br J Haematol. 2020 Sep 30.
      Treatment of relapsed/resistant acute myeloid leukaemia (AML) remains a significant area of unmet patient need, the outlook for most patients remaining extremely poor. A promising approach is to augment the anti-tumour immune response in these patients; most cancers do not activate immune effector cells because they express immunosuppressive ligands. We have previously shown that CD200 (an immunosuppressive ligand) is overexpressed in AML and confers an inferior overall survival compared to CD200low/neg patients. Here we show that a fully human anti-CD200 antibody (TTI-CD200) can block the interaction of CD200 with its receptor and restore AML immune responses in vitro and in vivo.
    Keywords:  AML; CD200; immune check point; immunosuppression; smouldering multiple myeloma
  18. J Clin Med. 2020 Sep 25. pii: E3090. [Epub ahead of print]9(10):
      Acute myeloid leukemia (AML) is a hematologic malignancy characterized by the rapid and uncontrolled clonal growth of myeloid lineage cells in the bone marrow. The advent of oral, selective inhibitors of the B-cell leukemia/lymphoma-2 (BCL-2) apoptosis pathway, such as venetoclax, will likely induce a paradigm shift in the treatment of AML. However, the high cost of this treatment and the risk of additive toxicity when used in combination with standard chemotherapy represent limitations to its use and underscore the need to identify which patients are most-and least-likely to benefit from incorporation of venetoclax into the treatment regimen. Bone marrow specimens from 93 newly diagnosed AML patients were collected in this study and evaluated for BCL-2 protein expression by immunohistochemistry. Using this low-cost, easily, and readily applicable analysis method, we found that 1 in 5 AML patients can be considered as BCL-2-. In addition to a lower bone marrow blast percentage, this group exhibited a favorable molecular profile characterized by lower WT1 expression and underrepresentation of FLT3 mutations. As compared to their BCL-2+ counterparts, the absence of BCL-2 expression was associated with a favorable response to standard chemotherapy and overall survival, thus potentially precluding the necessity for venetoclax add-on.
    Keywords:  BCL-2; FLT3; acute myeloid leukemia; immunohistochemistry; next-generation sequencing; venetoclax