bims-tremyl Biomed News
on Therapy resistance biology in myeloid leukemia
Issue of 2022‒07‒31
thirty-two papers selected by
Paolo Gallipoli
Barts Cancer Institute, Queen Mary University of London

  1. Cancer Discov. 2022 Jul 29. pii: CD-21-1631. [Epub ahead of print]
      Recently, screens for mediators of resistance to FLT3 and ABL kinase inhibitors in leukemia resulted in the discovery of LZTR1 as an adaptor of a Cullin-3 RING E3 ubiquitin ligase complex responsible for degradation of RAS GTPases. In parallel, dysregulated LZTR1 expression via aberrant splicing and mutations were identified in clonal hematopoietic conditions. Here we identify that loss of LZTR1, or leukemia-associated mutants in the LZTR1 substrate and RAS GTPase RIT1 which escape degradation, drive hematopoietic stem cell (HSC) expansion and leukemia in vivo. While RIT1 stabilization was sufficient to drive hematopoietic transformation, transformation mediated by LZTR1 loss required MRAS. RAS targeting bioPROTACs or reduction of GTP-loaded RAS overcomes LZTR1 loss-mediated resistance to FLT3 inhibitors. These data reveal proteolysis of non-canonical RAS proteins as novel regulators of HSC self-renewal, define the function of RIT1 and LZTR1 mutations in leukemia, and identify means to overcome drug resistance due to LZTR1 downregulation.
  2. Cancers (Basel). 2022 Jul 15. pii: 3456. [Epub ahead of print]14(14):
      Venetoclax is a BCL-2 inhibitor that effectively improves clinical outcomes in newly diagnosed, relapsed and refractory acute myeloid leukemia (AML) patients, with complete response rates (with and without complete blood count recovery) ranging between 34-90% and 21-33%, respectively. Here, we aim to give an overview of the efficacy of venetoclax-based therapy for AML patients, as compared to standard chemotherapy, and on factors and mechanisms involved in venetoclax sensitivity and resistance in AML (stem) cells, with the aim to obtain a perspective of response biomarkers and combination therapies that could enhance the sensitivity of AML cells to venetoclax. The presence of molecular aberrancies can predict responses to venetoclax, with a higher response in NPM1-, IDH1/2-, TET2- and relapsed or refractory RUNX1-mutated AML. Decreased sensitivity to venetoclax was observed in patients harboring FLT3-ITD, TP53, K/NRAS or PTPN11 mutations. Moreover, resistance to venetoclax was observed in AML with a monocytic phenotype and patients pre-treated with hypomethylating agents. Resistance to venetoclax can arise due to mutations in BCL-2 or pro-apoptotic proteins, an increased dependency on MCL-1, and usage of additional/alternative sources for energy metabolism, such as glycolysis and fatty acid metabolism. Clinical studies are testing combination therapies that may circumvent resistance, including venetoclax combined with FLT3- and MCL-1 inhibitors, to enhance venetoclax-induced cell death. Other treatments that can potentially synergize with venetoclax, including MEK1/2 and mitochondrial complex inhibitors, need to be evaluated in a clinical setting.
    Keywords:  AML; biomarkers; resistance; sensitivity; therapeutic combinations; venetoclax (BCL-2 inhibitor)
  3. Cancers (Basel). 2022 Jul 13. pii: 3398. [Epub ahead of print]14(14):
      The treatment of many types of cancers, including acute myeloid leukemia (AML), has been revolutionized by the development of therapeutics targeted at crucial molecular drivers of oncogenesis. In contrast to broad, relatively indiscriminate conventional chemotherapy, these targeted agents precisely disrupt key pathways within cancer cells. FMS-like tyrosine kinase 3 (FLT3)-encoding a critical regulator of hematopoiesis-is the most frequently mutated gene in patients with AML, and these mutations herald reduced survival and increased relapse in these patients. Approximately 30% of newly diagnosed AML carries an FLT3 mutation; of these, approximately three-quarters are internal tandem duplication (ITD) mutations, and the remainder are tyrosine kinase domain (TKD) mutations. In contrast to its usual, tightly controlled expression, FLT3-ITD mutants allow constitutive, "run-away" activation of a large number of key downstream pathways which promote cellular proliferation and survival. Targeted inhibition of FLT3 is, therefore, a promising therapeutic avenue. In April 2017, midostaurin became both the first FLT3 inhibitor and the first targeted therapy of any kind in AML to be approved by the US FDA. The use of FLT3 inhibitors has continued to grow as clinical trials continue to demonstrate the efficacy of this class of agents, with an expanding number available for use as both experimental standard-of-care usage. This review examines the biology of FLT3 and its downstream pathways, the mechanism of FLT3 inhibition, the development of the FLT3 inhibitors as a class and uses of the agents currently available clinically, and the mechanisms by which resistance to FLT3 inhibition may both develop and be overcome.
    Keywords:  AML; FLT3; FLT3 inhibitor; acute myeloid leukemia; crenolanib; gilteritinib; midostaurin; quizartinib; sorafenib
  4. Blood. 2022 Jul 27. pii: blood.2022016033. [Epub ahead of print]
      We have developed a deep-scale proteome and phosphoproteome database from 44 representative Acute Myeloid Leukemia (AML) patients from the LAML TCGA dataset, and 6 healthy bone marrow derived controls. After confirming data quality, we orthogonally validated several previously undescribed features of AML revealed by the proteomic data. We identified examples of post-transcriptionally regulated proteins both globally (i.e. in all AML samples), and also, in patients with recurrent AML driver mutations. For example, samples with IDH1/2 mutations displayed elevated levels of the 2‑oxoglutarate-dependent histone demethylases KDM4A/B/C, despite no changes in mRNA levels for these genes; we confirmed this finding in vitro. In samples with NPMc mutations, we identified several nuclear importins with post‑transcriptionally increased protein abundance, and showed that they interact with NPMc, but not wildtype NPM1. We identified two cell surface proteins (CD180 and MRC1/CD206) expressed on AML blasts of many patients (but not healthy CD34+ stem/progenitor cells) that could represent novel targets for immunologic therapies, and confirmed these targets via flow cytometry. Finally, we detected nearly 30,000 phosphosites in these samples; globally, AML samples were associated with the abnormal phosphorylation of specific residues in PTPN11, STAT3, AKT1 and PRKCD. FLT3‑TKD samples were associated with increased phosphorylation of activating tyrosines on the cytoplasmic Src-family tyrosine kinases FGR and HCK, and related signaling proteins. PML-RARA-initiated AML samples displayed a unique phosphorylation signature, and TP53-mutant samples showed abundant phosphorylation of serine-183 on TP53 itself. This publicly available database will serve as a foundation for further investigations of protein dysregulation in AML pathogenesis.
  5. J Clin Invest. 2022 Jul 28. pii: e152673. [Epub ahead of print]
      Myelodysplastic syndromes (MDS) are age-related myeloid neoplasms with increased risks of progression to acute myeloid leukemia (AML). The mechanisms of MDS to AML transformation are poorly understood, especially in relation to the aging microenvironment. We previously established a mDia1/miR-146a double knockout (DKO) mouse model phenocopying MDS. These mice develop age-related pancytopenia with over-secretion of pro-inflammatory cytokines. Here, we found that most of the DKO mice underwent leukemic transformation at 12-14 months of age. These mice showed myeloblast replacement of a fibrotic bone marrow and widespread leukemic infiltration. Strikingly, depletion of IL-6 in these mice largely rescued the leukemic transformation and markedly extended the survival. Single cell RNA sequencing analyses revealed that DKO leukemic mice had increased monocytic blasts that were reduced with IL-6 knockout. We further revealed that the levels of surface and soluble IL-6 receptor (IL-6R) in the bone marrow were significantly increased in high risk MDS patients. Similarly, IL-6R was also highly expressed in older DKO mice. Blocking of IL-6 signaling significantly ameliorated AML progression in the DKO model and clonogenicity of CD34 positive cells from MDS patients. Our study establishes a mouse model of age-related MDS to AML progression and indicates the clinical significance of targeting IL-6 signaling in treating high risk MDS.
    Keywords:  Cancer; Hematology; Inflammation; Mouse models
  6. Cancers (Basel). 2022 Jul 22. pii: 3567. [Epub ahead of print]14(15):
      Novel therapeutic tools are warranted to improve outcomes for children with acute myeloid leukemia (AML). Differences in the proteome of leukemic blasts and stem cells (AML-SCs) in AML compared with normal hematopoietic stem cells (HSCs) may facilitate the identification of potential targets for future treatment strategies. In this explorative study, we used mass spectrometry to compare the proteome of AML-SCs and CLEC12A+ blasts from five pediatric AML patients with HSCs and hematopoietic progenitor cells from hematologically healthy, age-matched controls. A total of 456 shared proteins were identified in both leukemic and control samples. Varying protein expression profiles were observed in AML-SCs and leukemic blasts, none having any overall resemblance to healthy counterpart cell populations. Thirty-four proteins were differentially expressed between AML-SCs and HSCs, including the upregulation of HSPE1, SRSF1, and NUP210, and the enrichment of proteins suggestive of protein synthesis perturbations through the downregulation of EIF2 signaling was found. Among others, NUP210 and calreticulin were upregulated in CLEC12A+ blasts compared with HSCs. In conclusion, the observed differences in protein expression between pediatric patients with AML and pediatric controls, in particular when comparing stem cell subsets, encourages the extended exploration of leukemia and AML-SC-specific biomarkers of potential relevance in the development of future therapeutic options in pediatric AML.
    Keywords:  hematopoietic stem cells; mass spectrometry; pediatric acute myeloid leukemia; proteomics
  7. Mol Cancer Res. 2022 Jul 28. pii: MCR-22-0109. [Epub ahead of print]
      NRAS proteins are central regulators of proliferation, survival, and self-renewal in leukemia. Previous work demonstrated that the effects of oncogenic NRAS in mediating proliferation and self-renewal are mutually exclusive within leukemia subpopulations and that levels of oncogenic NRAS vary between highly proliferative and self-renewing leukemia subpopulations. These findings suggest that NRAS activity levels may be important determinants of leukemic behavior. To define how oncogenic NRAS levels affect these functions, we genetically engineered an acute myeloid leukemia (AML) cell line, THP¬-1, to express variable levels of NRASG12V. We replaced the endogenous NRASG12D gene with a tetracycline-inducible and dose-responsive NRASG12V transgene. Cells lacking NRASG12V oncoprotein were cell cycle arrested. Intermediate levels of NRASG12V induced maximal proliferation; higher levels led to attenuated proliferation, increased G1 arrest, senescence markers, and maximal self-renewal capacity. Higher levels of the oncoprotein also induced self-renewal and mitochondrial genes. We used mass cytometry (CyTOF) to define the downstream signaling events that mediate these differential effects. Not surprisingly, we found that the levels of such canonical RAS-effectors as pERK and p4EBP1 correlated with NRASG12V levels. Beta-catenin, a mediator of self-renewal, also correlated with NRASG12V levels. These signaling intermediates may mediate the differential effects of NRASG12V in leukemia biology. Together, these data reveal that oncogenic NRAS levels are important determinants of leukemic behavior explaining heterogeneity in phenotypes within a clone. This system provides a new model to study RAS oncogene addiction and RAS-induced self-renewal in AML. Implications: Different levels of activated NRAS may exert distinct effects on proliferation and self-renewal.
  8. Clin Proteomics. 2022 Jul 27. 19(1): 30
      Acute Myeloid Leukemia (AML) affects 20,000 patients in the US annually with a five-year survival rate of approximately 25%. One reason for the low survival rate is the high prevalence of clonal evolution that gives rise to heterogeneous sub-populations of leukemic cells with diverse mutation spectra, which eventually leads to disease relapse. This genetic heterogeneity drives the activation of complex signaling pathways that is reflected at the protein level. This diversity makes it difficult to treat AML with targeted therapy, requiring custom patient treatment protocols tailored to each individual's leukemia. Toward this end, the Beat AML research program prospectively collected genomic and transcriptomic data from over 1000 AML patients and carried out ex vivo drug sensitivity assays to identify genomic signatures that could predict patient-specific drug responses. However, there are inherent weaknesses in using only genetic and transcriptomic measurements as surrogates of drug response, particularly the absence of direct information about phosphorylation-mediated signal transduction. As a member of the Clinical Proteomic Tumor Analysis Consortium, we have extended the molecular characterization of this cohort by collecting proteomic and phosphoproteomic measurements from a subset of these patient samples (38 in total) to evaluate the hypothesis that proteomic signatures can improve the ability to predict response to 26 drugs in AML ex vivo samples. In this work we describe our systematic, multi-omic approach to evaluate proteomic signatures of drug response and compare protein levels to other markers of drug response such as mutational patterns. We explore the nuances of this approach using two drugs that target key pathways activated in AML: quizartinib (FLT3) and trametinib (Ras/MEK), and show how patient-derived signatures can be interpreted biologically and validated in cell lines. In conclusion, this pilot study demonstrates strong promise for proteomics-based patient stratification to assess drug sensitivity in AML.
  9. Leukemia. 2022 Jul 28.
      Gene mutations independent of BCR::ABL1 have been identified in newly diagnosed patients with chronic myeloid leukemia (CML) in chronic phase, whereby mutations in epigenetic modifier genes were most common. These findings prompted the systematic analysis of prevalence, dynamics, and prognostic significance of such mutations, in a clinically well-characterized patient population of 222 CML patients from the TIGER study (CML-V) by targeted next-generation sequencing covering 54 myeloid leukemia-associated genes. In total, 53/222 CML patients (24%) carried 60 mutations at diagnosis with ASXL1 being most commonly affected (n = 20). To study mutation dynamics, longitudinal deep sequencing analysis of serial samples was performed in 100 patients after 12, 24, and 36 months of therapy. Typical patterns of clonal evolution included eradication, persistence, and emergence of mutated clones. Patients carrying an ASXL1 mutation at diagnosis showed a less favorable molecular response to nilotinib treatment, as a major molecular response (MMR) was achieved less frequently at month 12, 18, and 24 compared to all other patients. Patients with ASXL1 mutations were also younger and more frequently found in the high risk category, suggesting a central role of clonal evolution associated with ASXL1 mutations in CML pathogenesis.
  10. Cancers (Basel). 2022 Jul 11. pii: 3375. [Epub ahead of print]14(14):
      Acute myeloid leukemia (AML) with ≥2% plasmacytoid dendritic cells (pDC) has been recently described as AML with pDC differentiation (pDC-AML) characterized by pDC expansion with frequent RUNX1 mutations. In this study, we investigated a cohort of 53 pDC-AML cases representing about 3% of all AML cases. We characterized their immunophenotype and genetic profiles and compared these findings with blastic plasmacytoid dendritic cell neoplasm (BPDCN). pDC-differentiation/expansion was preferentially observed in AML with an immature myeloid or myelomonocytic immunophenotype, where myeloblasts were frequently positive for CD34 (98%), CD117 (94%), HLA-DR (100%) and TdT (79%), with increased CD123 (89%) expression. The median number of pDCs in pDC-AML was 6.6% (range, 2% to 26.3%) and their immunophenotype reminiscent of pDCs in early or intermediate stages of differentiation. The immunophenotype of pDCs in pDC-AML was different from BPDCN (n = 39), with major disparities in CD34 (96% vs. 0%), CD56 (8% vs. 97%) and TCL1 (12% vs. 98%) and significant differences in frequency of CD4, CD13, CD22, CD25, CD36, CD38, CD117 and CD303 expression. At the molecular level, the genetic landscapes of pDC-AML and BPDCN also differ, with RUNX1 mutations detected in 64% of pDC-AML versus 2% of BPDCN. Disparities in TET2 (21% vs. 56%), FLT3 (23% vs. 0%), DNMT3A (32% vs. 10%) and ZRSR2 (2% vs. 16%) (all p < 0.05) were also detected. The distinct immunophenotypic and mutation profiles of pDC-AML and BPDCN indicate that the neoplastic pDCs in pDC-AML and BPDCN derived from different subsets of pDC precursors.
    Keywords:  BPDCN; acute myeloid leukemia; flow cytometry; immunophenotype; mutation; plasmacytoid dendritic cells
  11. Cells. 2022 Jul 20. pii: 2249. [Epub ahead of print]11(14):
      Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of many solid tumors, with limited progress made in the area of myeloid malignancies. The low mutational burden of acute myeloid leukemia (AML) is one potential reason behind the lack of activity of T-cell harnessing ICIs, particularly CTLA-4 and PD-1 inhibitors. Innate immune checkpoints play a critical role in the immune escape of AML and myelodysplastic syndromes (MDS). The CD47 targeting agent, magrolimab, has shown promising activity when combined with azacitidine in early phase trials conducted in AML and higher-risk MDS, especially among patients harboring a TP53 mutation. Similarly, sabatolimab (an anti-TIM-3 monoclonal antibody) plus hypomethylating agents have shown durable responses in higher-risk MDS and AML in early clinical trials. Randomized trials are currently ongoing to confirm the efficacy of these agents. In this review, we will present the current progress and future directions of immune checkpoint inhibition in AML and MDS.
    Keywords:  CD47; TIM-3; acute myeloid leukemia; immune checkpoint inhibitors; myelodysplastic syndromes
  12. Ther Adv Hematol. 2022 ;13 20406207221112899
      Myelodysplastic syndrome (MDS) is a clonal hematopoietic stem cell disorder clinically defined by cytopenias, bone marrow failure, and an increased risk of progressing to acute myeloid leukemia (AML). Traditionally, first-line treatment for patients with higher-risk MDS has been hypomethylating agents (HMAs). However, these agents have modest clinical activity as single agents. A one-size-fits-all treatment paradigm is insufficient for such a heterogeneous disease in the modern era of precision medicine. Several new agents have been developed for MDS with the hopes of improving clinical outcomes and survival. Pevonedistat is a first-in-class, novel inhibitor of neuronal precursor cell-expressed developmentally down-regulated protein-8 (NEDD8) activating enzyme (NAE) blocking the neddylation pathway leading to downstream effects on the ubiquitin-proteosome pathway. Pevonedistat ultimately leads to apoptosis and inhibition of the cell cycle in cancer cells. Studies have demonstrated the safety profile of pevonedistat, leading to the development of multiple trials investigating combination strategies with pevonedistat in MDS and AML. In this review, we summarize the preclinical and clinical rationale for pevonedistat in MDS and AML, review the clinical data of this agent alone and in combination with HMAs to date, and highlight potential future directions for this agent in myeloid malignancies.
    Keywords:  Acute myeloid leukemia (AML); NEDD8 activating enzyme (NAE) inhibitor; myelodysplastic syndrome (MDS); pevonedistat
  13. Haematologica. 2022 Jul 28.
      The outcome of acute myeloid leukemia (AML) remains poor, and immunotherapy has the potential to improve this. T cells expressing chimeric antigen receptors (CARs) or bispecific T cell engagers targeting CD123 are actively being explored in preclinical and/or early phase clinical studies. We have shown that T cells expressing CD123-specific bispecific T cell engagers (CD123.ENG T cells) have anti-AML activity. However, like CAR T cells, their effector function diminishes rapidly once they are repeatedly exposed to antigenpositive target cells. Here we sought to improve the effector function of CD123.ENG T cells by expressing inducible costimulatory molecules consisting of MyD88 and CD40 (iMC), MyD88 (iM), or CD40 (iC), which are activated by a chemical inducer of dimerization (CID). CD123.ENG T cells expressing iMC, iM, or iC maintained their antigen specificity in the presence of CID as judged by cytokine production (IFNc, IL-2) and their cytolytic activity. In repeat stimulation assays, activating iMC and iM, in contrast to iC, enabled CD123.ENG T cells to secrete cytokines, expand, and kill CD123-positive target cells repeatedly. Activating iMC in CD123.ENG T cells consistently improved anti-tumor activity in an AML xenograft model. This translated into a significant survival advantage in comparison to mice that received CD123.ENG or CD123.ENG.iC T cells. In contrast, activation of only iM in CD123.ENG T cells resulted in donor-dependent antitumor activity. Our work highlights the need for both toll-like receptor (TLR) pathway activation via MyD88 and provision of costimulation via CD40 to consistently enhance the antitumor activity of CD123.ENG T cells.
  14. Cancers (Basel). 2022 Jul 22. pii: 3576. [Epub ahead of print]14(15):
      The prognostic value of measurable residual disease (MRD) by flow cytometry in acute myeloid leukemia (AML) patients treated with non-intensive therapy is relatively unexplored. The clinical value of MRD threshold below 0.1% is also unknown after non-intensive therapy. In this study, MRD to a sensitivity of 0.01% was analyzed in sixty-three patients in remission after azacitidine/venetoclax treatment. Multivariable cox regression analysis identified prognostic factors associated with cumulative incidence of relapse (CIR), progression-free survival (PFS) and overall survival (OS). Patients who achieved MRD < 0.1% had a lower relapse rate than those who were MRD ≥ 0.1% at 18 months (13% versus 57%, p = 0.006). Patients who achieved an MRD-negative CR had longer median PFS and OS (not reached and 26.5 months) than those who were MRD-positive (12.6 and 10.3 months, respectively). MRD < 0.1% was an independent predictor for CIR, PFS, and OS, after adjusting for European Leukemia Net (ELN) risk, complex karyotype, and transplant (HR 5.92, 95% CI 1.34-26.09, p = 0.019 for PFS; HR 2.60, 95% CI 1.02-6.63, p = 0.046 for OS). Only an MRD threshold of 0.1%, and not 0.01%, was predictive for OS. Our results validate the recommended ELN MRD cut-off of 0.1% to discriminate between patients with improved CIR, PFS, and OS after azacitidine/venetoclax therapy.
    Keywords:  acute myeloid leukemia; measurable residual disease; venetoclax
  15. Leukemia. 2022 Jul 29.
      Epigenetic modifications have been found to play crucial roles in myelodysplastic neoplasm (MDS) progression. Previously, we investigated genome-wide DNA methylation alterations during MDS evolution to acute myeloid leukemia (AML) by next-generation sequencing (NGS). Herein, we further determined the role and clinical implications of an evident methylation change in CpG islands at the SLIT2 promoter identified by NGS. First, increased SLIT2 promoter methylation was validated in 11 paired MDS/AML patients during disease evolution. Additionally, SLIT2 promoter methylation was markedly increased in MDS/AML patients compared with controls and was correlated with poor clinical phenotype and outcome. Interestingly, SLIT2 expression was particularly upregulated in AML patients and was not correlated with SLIT2 promoter methylation. However, the SLIT2-embedded genes SLIT2-IT1 and miR-218 were downregulated in AML patients, which was negatively associated with SLIT2 promoter methylation and further validated by demethylation studies. Functionally, SLIT2-IT1/miR-218 overexpression exhibited antileukemic effects by affecting cell proliferation, apoptosis and colony formation in vitro and in vivo. Mechanistically, SLIT2-IT1 may function as a competing endogenous RNA by sponging miR-3156-3p to regulate BMF expression, whereas miR-218 may directly target HOXA1 in MDS progression. In summary, our findings demonstrate that SLIT2 promoter hypermethylation is associated with disease evolution in MDS and predicts poor prognoses in both MDS and AML. Epigenetic inactivation of SLIT2-IT1/miR-218 by SLIT2 promoter hypermethylation could be a promising therapeutic target in MDS.
  16. Am J Hematol. 2022 Jul 24.
      Azacitidine and decitabine are hypomethylating agents that have dose-dependent epigenetic and cytotoxic effects and are widely used in the treatment of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). In this review, we discuss the path to regulatory approval of azacitidine and decitabine, highlighting the substantial efforts that have been made to optimize the dosing schedule and administration of these drugs, including the development of new, oral formulations of both agents. We also review novel combination strategies that are being investigated in ongoing clinical trials for patients with MDS and AML, as well as efforts to expand the current indications of these agents. This article is protected by copyright. All rights reserved.
  17. Blood. 2022 Jul 25. pii: blood.2022016194. [Epub ahead of print]
      Historically, Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) has been associated with poor outcomes and allogeneic hematopoietic cell transplantation (allo-HCT) is recommended in first complete remission (CR1). However, in the tyrosine kinase inhibitor (TKI) era, rapid attainment of a complete molecular remission (CMR) is associated with excellent outcomes without allo-HCT, suggesting transplant may not be required for these patients. To test this hypothesis, we retrospectively identified adult patients with Ph+ ALL treated with induction therapy including TKIs and attained CMR within 90 days of diagnosis at 5 transplant centers in the United States. We compared outcomes of those who did and did not receive allo-HCT in first remission. We identified 230 patients (Allo-HCT: 98, non-HCT: 132). The allo-HCT cohort was younger with better performance status. On multivariable analysis (MVA), allo-HCT was not associated with improved overall survival (aHR 1.05, 95% C.I. 0.63 - 1.73) or relapse-free survival (aHR: 0.86, 95% C.I. 0.54 - 1.37) compared to non-HCT treatment. Allo-HCT was associated with a lower cumulative incidence of relapse (aHR 0.32, 95% C.I. 0.17 - 0.62) but higher non-relapse mortality (aHR: 2.59, 95% C.I. 1.37 - 4.89). Propensity score matching analysis confirmed results of MVA. Comparison of reduced-intensity HCT to non-HCT showed no statistically significant difference in any of the above endpoints. In conclusion, adult patients with Ph+ ALL who achieved CMR within 90 days of starting treatment did not derive a survival benefit from allo-HCT in CR1 in this retrospective study.
  18. Nat Commun. 2022 Jul 23. 13(1): 4267
      Mutations in genes that confer a selective advantage to hematopoietic stem cells (HSCs) drive clonal hematopoiesis (CH). While some CH drivers have been identified, the compendium of all genes able to drive CH upon mutations in HSCs remains incomplete. Exploiting signals of positive selection in blood somatic mutations may be an effective way to identify CH driver genes, analogously to cancer. Using the tumor sample in blood/tumor pairs as reference, we identify blood somatic mutations across more than 12,000 donors from two large cancer genomics cohorts. The application of IntOGen, a driver discovery pipeline, to both cohorts, and more than 24,000 targeted sequenced samples yields a list of close to 70 genes with signals of positive selection in CH, available at . This approach recovers known CH genes, and discovers other candidates.
  19. Nucleic Acids Res. 2022 Jul 25. pii: gkac613. [Epub ahead of print]
      Although originally described as transcriptional activator, SPI1/PU.1, a major player in haematopoiesis whose alterations are associated with haematological malignancies, has the ability to repress transcription. Here, we investigated the mechanisms underlying gene repression in the erythroid lineage, in which SPI1 exerts an oncogenic function by blocking differentiation. We show that SPI1 represses genes by binding active enhancers that are located in intergenic or gene body regions. HDAC1 acts as a cooperative mediator of SPI1-induced transcriptional repression by deacetylating SPI1-bound enhancers in a subset of genes, including those involved in erythroid differentiation. Enhancer deacetylation impacts on promoter acetylation, chromatin accessibility and RNA pol II occupancy. In addition to the activities of HDAC1, polycomb repressive complex 2 (PRC2) reinforces gene repression by depositing H3K27me3 at promoter sequences when SPI1 is located at enhancer sequences. Moreover, our study identified a synergistic relationship between PRC2 and HDAC1 complexes in mediating the transcriptional repression activity of SPI1, ultimately inducing synergistic adverse effects on leukaemic cell survival. Our results highlight the importance of the mechanism underlying transcriptional repression in leukemic cells, involving complex functional connections between SPI1 and the epigenetic regulators PRC2 and HDAC1.
  20. Stem Cell Res. 2022 Jul 09. pii: S1873-5061(22)00210-0. [Epub ahead of print]63 102861
      Acute promyelocytic leukemia (APL) M3 is an acute myeloid leukemia (AML) subtype and is characterized by the chromosomal translocation t(15;17)(p22;q11), which results in the fusion of the promyelocytic gene (PML) at 15q22 with the retinoic acid α-receptor gene (RARA) at 17q21. We generated an induced pluripotent stem cell line "KUMi003-A" from an APL M3 patient that is pluripotent and can differentiate into the three germ layers. This iPSC line will be useful as a disease model to investigate disease mechanisms specific to APL M3.
  21. Leukemia. 2022 Jul 27.
      Predictors, genetic characteristics, and long-term outcomes of patients with SAA who clonally evolved after immunosuppressive therapy (IST) were assessed. SAA patients were treated with IST from 1989-2020. Clonal evolution was categorized as "high-risk" (overt myeloid neoplasm [meeting WHO criteria for dysplasia, MPN or acute leukemia] or isolated chromosome-7 abnormality/complex karyotype without dysplasia or overt myeloid neoplasia) or "low-risk" (non-7 or non-complex chromosome abnormalities without morphological evidence of dysplasia or myeloid neoplasia). Univariate and multivariate analysis using Fine-Gray competing risk regression model determined predictors. Long-term outcomes included relapse, overall survival (OS) and hematopoietic stem cell transplant (HSCT). Somatic mutations in myeloid cancer genes were assessed in evolvers and in 407 patients 6 months after IST. Of 663 SAA patients, 95 developed clonal evolution. Pre-treatment age >48 years and ANC > 0.87 × 109/L were strong predictors of high-risk evolution. OS was 37% in high-risk clonal evolution by 5 years compared to 94% in low-risk. High-risk patients who underwent HSCT had improved OS. Eltrombopag did not increase high-risk evolution. Splicing factors and RUNX1 somatic variants were detected exclusively at high-risk evolution; DNMT3A, BCOR/L1 and ASXL1 were present in both. RUNX1, splicing factors and ASXL1 somatic mutations detected at 6 months after IST predicted high-risk evolution.
  22. Blood. 2022 Jun 25. pii: blood.2021014612. [Epub ahead of print]
      Avapritinib, a selective inhibitor of KIT D816V, was approved by the Food and Drug Administration in 2021 for treatment of advanced systemic mastocytosis (AdvSM), and by the European Medicines Agency in 2022 for AdvSM patients after prior systemic therapy. The phase I EXPLORER and phase II PATHFINDER trials demonstrated that avapritinib can elicit complete and durable clinical responses, and molecular remission of KIT D816V. Key management challenges relate to the complex mutational landscape of AdvSM, often found with an associated hematologic neoplasm.
  23. Biomedicines. 2022 Jun 28. pii: 1530. [Epub ahead of print]10(7):
      Hematopoietic stem cell transplantation (HSCT) is a curative post-remission treatment in patients with acute myeloid leukemia (AML), but relapse after transplant is still a challenging event. In recent year, several studies have investigated the molecular minimal residual disease (qPCR-MRD) as a predictor of relapse, but the lack of standardized protocols, cut-offs, and timepoints, especially in the pediatric setting, has prevented its use in several settings, including before HSCT. Here, we propose the first collaborative retrospective I-BFM-AML study assessing qPCR-MRD values in pretransplant bone marrow samples of 112 patients with a diagnosis of AML harboring t(8;21)(q22; q22)RUNX1::RUNX1T1, or inv(16)(p13q22)CBFB::MYH11, or t(9;11)(p21;q23)KMT2A::MLLT3, or FLT3-ITD genetic markers. We calculated an ROC cut-off of 2.1 × 10-4 that revealed significantly increased OS (83.7% versus 57.1%) and EFS (80.2% versus 52.9%) for those patients with lower qPCR-MRD values. Then, we partitioned patients into three qPCR-MRD groups by combining two different thresholds, 2.1 × 10-4 and one lower cut-off of 1 × 10-2, and stratified patients into low-, intermediate-, and high-risk groups. We found that the 5-year OS (83.7%, 68.6%, and 39.2%, respectively) and relapse-free survival (89.2%, 73.9%, and 67.9%, respectively) were significantly different independent of the genetic lesion, conditioning regimen, donor, and stem cell source. These data support the PCR-based approach playing a clinical relevance in AML transplant management.
    Keywords:  AML; HSCT; MRD; molecular genetics; q-PCR
  24. Blood. 2022 Jul 26. pii: blood.2022016112. [Epub ahead of print]
      Hematopoietic stem cells (HSCs) have reduced capacities to properly maintain and replenish the hematopoietic system during myelosuppressive injury or aging. Expanding and rejuvenating HSCs for therapeutic purposes has been a long-sought goal, with limited progress. Here, we show that enzyme sphingosine kinase 2 (Sphk2), which generates the lipid metabolite sphingosine-1-phosphate, is highly expressed in HSCs. The deletion of Sphk2 markedly promotes self-renewal and increases the regenerative potential of HSCs. More importantly, Sphk2 deletion globally preserves the young HSC gene expression pattern, improves the function, and sustains the multilineage potential of HSCs during aging. Mechanistically, Sphk2 interacts with prolyl hydroxylase 2 and the Von Hippel-Lindau protein to facilitate HIF1α ubiquitination in the nucleus independent of the Sphk2 catalytic activity. Deletion of Sphk2 increases hypoxic responses by stabilizing the HIF1α protein to upregulate PDK3, a glycolysis checkpoint protein for HSC quiescence, which subsequently enhances the function of HSCs by improving their metabolic fitness; specifically, it enhances anaerobic glycolysis but suppresses mitochondrial oxidative phosphorylation and generation of reactive oxygen species. Overall, targeting Sphk2 to enhance the metabolic fitness of HSCs is a promising strategy to expand and rejuvenate functional HSCs.
  25. Expert Rev Hematol. 2022 Jul 28.
      BACKGROUND: : Acute myeloid leukemia (AML) is a hematologic malignancy with genetic alterations. RUNX1, which is an essential transcription factor for hematopoiesis, is frequently mutated in AML. Loss of function mutation of RUNX1 is correlated to poor prognosis of AML patients. It is urgent to reveal the underlying mechanism.RESEARCH DESIGN AND METHODS: TCGA AML, GSE106291, GSE142700 and GSE67609 datasets were used. R package was used for define the differential expressed miRNAs, miRNA target genes, RUNX1 related gene, RUNX directly regulating genes, and so on. The relationship of gene expression with overall survival was analyzed by cox regression. KEGG and GO analysis were applied to the above mentioned genesets and overlapped genes. Alteration and importance of MAPK pathway was validated in K562 cells by Western blotting and apoptosis assay in vitro.
    RESULTS: RUNX1 regulated MAPK pathway indirectly and directly. MAPK pathway was altered in K562 cells induced mutated RUNX1, and these cells were more sensitive to AraC after p38 was inhibited.
    CONCLUSIONS: RUNX1 could modulate MAPK pathway, which may provide a potential therapeutic target for AML patients with RUNX1 mutations.
    Keywords:  AML; MAPK; RUNX1; mRNA; miRNA
  26. Transplant Cell Ther. 2022 Jul 25. pii: S2666-6367(22)01509-3. [Epub ahead of print]
      BACKGROUND: Allogeneic hematopoietic stem cell transplantation (SCT) after a patient with acute myeloid leukemia (AML) achieves a remission from intensive chemotherapy (IC) is given with curative intent. Recently, venetoclax-based regimens have become the standard of care for patients with newly-diagnosed AML who are unfit for IC. If these patients achieve remission, they may also be considered for potentially curative consolidation with SCT. There is limited data comparing outcomes following SCT after these different induction strategies.OBJECTIVE: The purpose of the current study was to evaluate depth of remission pre-SCT and outcomes post-SCT in adults with non-relapsed/refractory AML receiving pre-SCT therapy with either venetoclax/azacitidine (ven/aza) or IC.
    STUDY DESIGN: This was a retrospective, single-institution analysis of 169 patients receiving SCT in first remission after IC or ven/aza. Patient demographics and AML risk features were collected, as well as pre-SCT MRD assessed by flow cytometry and molecular methods. Relapse, transplant-related mortality, incidence of acute and chronic GVHD, and death from any cause were also recorded. Descriptive and survival statistics were applied to these data to compare IC and ven/aza groups. Cox proportional hazard models were utilized for univariate and multivariate analyses.
    RESULTS: We demonstrate that despite differences in baseline factors between these groups outcomes were similar. Relapse-free and overall survival as well as cumulative incidences (CI) of transplant-related mortality (TRM), relapse, and acute and chronic graft-versus-host-disease (GVHD) were comparable between groups. Exploring survival in younger (<65 years) versus older (>=65 years) patients by treatment group did not alter these results. Finally, although pre-SCT measurable residual disease (MRD) by flow cytometry was significantly predictive of post-SCT relapse and survival in the IC + SCT patients, it was not significantly predictive of relapse and survival in the ven/aza + SCT patients.
    CONCLUSIONS: While these findings require prospective validation in a larger cohort of patients, they suggest that ven/aza followed by SCT is a reasonable management strategy for transplant candidates at any age.
    Keywords:  AML; chemotherapy; mrd; survival; venetoclax
  27. Cancers (Basel). 2022 Jul 14. pii: 3431. [Epub ahead of print]14(14):
      Pathogenic loss-of-function RUNX1 germline variants cause autosomal dominantly-inherited familial platelet disorder with predisposition to hematologic malignancies (RUNX1-FPD). RUNX1-FPD is characterized by incomplete penetrance and a broad spectrum of clinical phenotypes, even within affected families. Heterozygous RUNX1 germline variants set the basis for leukemogenesis, but, on their own, they are not transformation-sufficient. Somatically acquired secondary events targeting RUNX1 and/or other hematologic malignancy-associated genes finally lead to MDS, AML, and rarely other hematologic malignancies including lymphoid diseases. The acquisition of different somatic variants is a possible explanation for the variable penetrance and clinical heterogeneity seen in RUNX1-FPD. However, individual effects of secondary variants are not yet fully understood. Here, we review 91 cases of RUNX1-FPD patients who predominantly harbor somatic variants in genes such as RUNX1, TET2, ASXL1, BCOR, PHF6, SRSF2, NRAS, and DNMT3A. These cases illustrate the importance of secondary events in the development and progression of RUNX1-FPD-associated hematologic malignancies. The leukemia-driving interplay of predisposing germline variants and acquired variants remain to be elucidated to better understand clonal evolution and malignant transformation and finally allow risk-adapted surveillance and targeted therapeutic measures to prevent leukemia.
    Keywords:  RUNX1 germline variants; RUNX1-FPD; hematologic malignancies; leukemia predisposition; somatic mutations
  28. Br J Haematol. 2022 Jul 28.
      Acute myeloid leukaemia (AML) is conventionally thought of as a medical emergency. However, several studies on the association of time from diagnosis to treatment with survival did not have concordant results. Here we analyse 55 985 AML patients from the National Cancer Database, and we show that in patients less than 60 years old a five-day delay in chemotherapy initiation leads to worse long-term survival. The difference is small [hazard ratio (HR) 1.05, 95% condidence interval (CI) 1.01-1.09 in multivariate analysis] but statistically significant. This study raises the issue of power to detect small differences in retrospective studies.
    Keywords:  acute myeloid leukaemia; time from diagnosis to treatment; time to treatment
  29. Haematologica. 2022 Jul 28.
      Elucidating genetic aberrations in pediatric acute myeloid leukemia (AML) provides insight in biology and may impact on risk-group stratification and clinical outcome. This study aimed to detect such aberrations in a selected series of samples without known (cyto)genetic aberration using molecular profiling. A cohort of 161 patients was selected from various study groups: DCOG, BFM, SJCRH, NOPHO and AEIOP. Samples were analyzed using RNA-sequencing (n=152), whole exome (n=135) and/or genome sequencing (WGS, n=100). In 70/156 patients (45%), of whom RNAseq or WGS was available, rearrangements were detected, 22 of which were novel; 5 involving ERGrearrangements and 4 NPM1-rearrangements. ERG-rearrangements showed self-renewal capacity in vitro, and a distinct gene expression pattern. Gene set enrichment analysis of this cluster showed upregulation of gene sets derived from Ewing sarcoma, which was confirmed comparing gene expression profiles of AML and Ewing sarcoma. Furthermore, NPM1-rearranged cases showed cytoplasmic NPM1 localization and revealed HOXA/B gene overexpression, as described for NPM1 mutated cases. Single-gene mutations as identified in adult AML were rare. Patients had a median of 24 coding mutations (range 7-159). Novel recurrent mutations were detected in UBTF (n=10), a regulator of RNA transcription. In 75% of patients an aberration with a prognostic impact could be detected. Therefore, we suggest these techniques need to become standard of care in diagnostics.
  30. Clin Cancer Res. 2022 Jul 25. pii: CCR-22-1618. [Epub ahead of print]
      PURPOSE: The molecular complexity of acute myeloid leukemia (AML) presents a considerable challenge to implementation of clinical genetic testing for accurate risk stratification. Identification of better biomarkers therefore remains a high priority to enable improving established stratification and guiding risk-adapted therapy decisions.EXPERIMENTAL DESIGN: We systematically integrated and analyzed the genome-wide CRISPR-Cas9 data from over 1,000 in vitro and in vivo knockout screens to identify the AML-specific fitness genes. A prognostic fitness score was developed using the sparse regression analysis in a training cohort of 618 cases and validated in five publicly available independent cohorts (n=1,570) and our RJAML cohort (n=157) with matched RNA-seq and targeted gene sequencing performed.
    RESULTS: A total of 280 genes were identified as AML fitness genes and a 16-gene AML fitness (AFG16) score was further generated and displayed highly prognostic power in more than 2,300 AML patients. The AFG16 score was able to distil downstream consequences of several genetic abnormalities and can substantially improve the European LeukemiaNet classification. The multi-omics data from the RJAML cohort further demonstrated its clinical applicability. Patients with high AFG16 scores had significantly poor response to induction chemotherapy. Ex vivo drug screening indicated that patients with high AFG16 scores were more sensitive to the cell cycle inhibitors, flavopiridol and SNS-032, and exhibited strongly activated cell cycle signaling.
    CONCLUSIONS: Our findings demonstrated the utility of the AFG16 score as a powerful tool for better risk stratification and selecting patients most likely to benefit from chemotherapy and alternative experimental therapies.
  31. Nature. 2022 Jul 27.
      In response to hormones and growth factors, the class I phosphoinositide-3-kinase (PI3K) signalling network functions as a major regulator of metabolism and growth, governing cellular nutrient uptake, energy generation, reducing cofactor production and macromolecule biosynthesis1. Many of the driver mutations in cancer with the highest recurrence, including in receptor tyrosine kinases, Ras, PTEN and PI3K, pathologically activate PI3K signalling2,3. However, our understanding of the core metabolic program controlled by PI3K is almost certainly incomplete. Here, using mass-spectrometry-based metabolomics and isotope tracing, we show that PI3K signalling stimulates the de novo synthesis of one of the most pivotal metabolic cofactors: coenzyme A (CoA). CoA is the major carrier of activated acyl groups in cells4,5 and is synthesized from cysteine, ATP and the essential nutrient vitamin B5 (also known as pantothenate)6,7. We identify pantothenate kinase 2 (PANK2) and PANK4 as substrates of the PI3K effector kinase AKT8. Although PANK2 is known to catalyse the rate-determining first step of CoA synthesis, we find that the minimally characterized but highly conserved PANK49 is a rate-limiting suppressor of CoA synthesis through its metabolite phosphatase activity. Phosphorylation of PANK4 by AKT relieves this suppression. Ultimately, the PI3K-PANK4 axis regulates the abundance of acetyl-CoA and other acyl-CoAs, CoA-dependent processes such as lipid metabolism and proliferation. We propose that these regulatory mechanisms coordinate cellular CoA supplies with the demands of hormone/growth-factor-driven or oncogene-driven metabolism and growth.
  32. Cancer Discov. 2022 Jul 29. pii: CD-21-1026. [Epub ahead of print]
      Chimeric Antigen Receptor T-cell (CART) immunotherapy led to unprecedented responses in patients with refractory/relapsed B-cell non-Hodgkin lymphoma (NHL); nevertheless, two-thirds of patients fail this treatment. Resistance to apoptosis is a key feature of cancer cells that associates with treatment failure. In 87 NHL patients treated with anti-CD19 CART, we found that chromosomal alteration of BCL-2, a critical anti-apoptotic regulator, in lymphoma cells was associated with reduced survival. Therefore, we combined CART19 with the FDA-approved BCL-2-inhibitor, venetoclax, and demonstrated in vivo synergy in venetoclax-sensitive NHL. However, higher venetoclax doses for venetoclax-resistant lymphomas resulted in CART toxicity. To overcome this limitation, we developed venetoclax-resistant CART by overexpressing mutated BCL-2(F104L) which is not recognized by venetoclax. Notably, BCL-2(F104L)-CART19 synergized with venetoclax in multiple lymphoma xenograft models. Furthermore, we uncovered that BCL-2 overexpression in T cells per se enhanced CART anti-tumor activity in preclinical models and in patients by prolonging CART persistence.