bims-tremyl Biomed News
on Therapy resistance biology in myeloid leukemia
Issue of 2022‒07‒10
24 papers selected by
Paolo Gallipoli
Barts Cancer Institute, Queen Mary University of London

  1. Blood. 2022 Jul 07. pii: blood.2022016867. [Epub ahead of print]
      The 2010 and 2017 editions of the European LeukemiaNet (ELN) recommendations for diagnosis and management of acute myeloid leukemia (AML) in adults are widely recognized among physicians and investigators. There have been major advances in our understanding of AML, including new knowledge about the molecular pathogenesis of AML, leading to an update of the disease classification, technological progress in genomic diagnostics and assessment of measurable residual disease, and the successful development of new therapeutic agents, such as FLT3, IDH1, IDH2, and BCL2 inhibitors. These advances have prompted this update which includes a revised ELN genetic risk classification, revised response criteria, and treatment recommendations.
  2. Blood Adv. 2022 Jul 07. pii: bloodadvances.2021005828. [Epub ahead of print]
      High Mobility Group AT-hook 2 (HMGA2) is a non-histone chromatin-binding protein which is normally expressed in stem cells of various tissues and aberrantly detected in several tumor types. We recently observed that one fourth of human Acute Myeloid Leukemia (AML) specimens express HMGA2, which associates with a very poor prognosis. We now present results indicating that HMGA2+ AMLs share a distinct transcriptional signature representing an immature phenotype. Using single cell analyses, we showed that HMGA2 is expressed in CD34+ subsets of stem cells and early progenitors, whether normal or derived from AML specimens. Of interest, we found that one of the strongest gene expression signatures associated with HMGA2 in AML is the upregulation of G2/M checkpoint genes. Whole genome CRISPR/Cas9 screening in HMGA2 overexpressing cells further revealed a synthetic lethal interaction with several G2/M checkpoint genes. Accordingly, small molecules that target G2/M proteins were preferentially active in vitro and in vivo on HMGA2+ AML specimens. Together, our findings suggest that HMGA2 is a key functional determinant in AML and is associated with stem cell features, G2/M status and related drug sensitivity.
  3. Nat Commun. 2022 Jul 04. 13(1): 3833
      Chemo-resistance in acute myeloid leukemia (AML) patients is driven by leukemic stem cells (LSCs) resulting in high rates of relapse and low overall survival. Here, we demonstrate that upregulation of the splicing factor, RBM17 preferentially marks and sustains LSCs and directly correlates with shorten patient survival. RBM17 knockdown in primary AML cells leads to myeloid differentiation and impaired colony formation and in vivo engraftment. Integrative multi-omics analyses show that RBM17 repression leads to inclusion of poison exons and production of nonsense-mediated decay (NMD)-sensitive transcripts for pro-leukemic factors and the translation initiation factor, EIF4A2. We show that EIF4A2 is enriched in LSCs and its inhibition impairs primary AML progenitor activity. Proteomic analysis of EIF4A2-depleted AML cells shows recapitulation of the RBM17 knockdown biological effects, including pronounced suppression of proteins involved in ribosome biogenesis. Overall, these results provide a rationale to target RBM17 and/or its downstream NMD-sensitive splicing substrates for AML treatment.
  4. Curr Opin Hematol. 2022 Jul 01. 29(4): 194-200
      PURPOSE OF REVIEW: Human aging is associated with an exponential increase in the occurrence of clonal hematopoiesis of indeterminate potential (CHIP). CHIP is associated with increased risks of de novo and therapy-related hematologic neoplasms and serves as a reservoir for leukemic relapse. Somatic mutations in the TP53 gene, which encodes the tumor suppressor protein p53, rank in the top five among genes that were mutated in CHIP. TP53 mutations in CHIP are associated with an increased incidence of myeloid neoplasms such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). This review focuses on mechanisms by which mutant p53 promotes CHIP progression and drives the pathogenesis of MDS and AML. We will also discuss potential therapeutic approaches that can target mutant p53 and improve treatment outcomes of MDS and AML.RECENT FINDINGS: TP53 was frequently mutated in individuals with CHIP as well as in patients with MDS and AML. While clinical studies suggest that p53 mutant hematopoietic stem and progenitor cell expansion may predispose the elderly to hematologic neoplasms, the underlying mechanisms are not fully understood. Recent findings suggest that mutant p53 may utilize both cell autonomous and noncell autonomous mechanisms to promote CHIP development. Furthermore, we and others have demonstrated that several gain-of-function mutant p53 proteins have enhanced oncogenic potential beyond dominant-negative and loss-of-function effects. Notably, TP53 allelic state has important implications for genome stability, clinical presentation, and outcomes in MDS. Some small molecules reactivating wild-type p53 tumor suppressor activity show promising effects on some human MDS and AML cells with TP53 mutations in preclinical and early phases of clinical studies.
    SUMMARY: TP53 mutations in MDS and AML are correlated with advanced disease, poor prognosis, reduced overall survival, and dismal outcomes. Deep understanding of the functions of mutant p53 proteins is essential to devise effective therapies for patients with myeloid neoplasms and other human cancers with TP53 mutations. Targeting mutant p53 directly or pathways regulated by mutant p53 holds great potential in preventing CHIP progression and treating MDS and AML patients with TP53 mutations.
  5. Blood Adv. 2022 Jul 07. pii: bloodadvances.2022007952. [Epub ahead of print]
      Despite the clinical benefit associated with gilteritinib in relapsed/refractory AML, most patients eventually develop resistance through unknown mechanisms. To delineate the mechanistic basis of resistance to gilteritinib, we performed targeted sequencing and scRNASeq on primary FLT3-ITD-mutated AML samples. Co-occurring mutations in RAS pathway genes were the most common genetic abnormalities, and unresponsiveness to gilteritinib was associated with increased expression of bone marrow-derived hematopoietic cytokines and chemokines. In particular, we found elevated expression of the TEK-family kinase, BMX, in gilteritinib-unresponsive patients pre- and post-treatment. BMX contributed to gilteritinib resistance in FLT3-mutant cell lines in a hypoxia-dependent manner by promoting pSTAT5 signaling, and these phenotypes could be reversed with pharmacological inhibition and genetic knockout. We also observed that inhibition of BMX in primary FLT3-mutated AML samples decreased chemokine secretion and enhanced the activity of gilteritinib. Subsequent gene module analyses indicated that gilteritinib responsiveness was associated with lymphocyte differentiation and myeloid leukocyte activation, whereas unresponsiveness to gilteritinib was associated with upregulation of cell-cycle, DNA/RNA metabolic processes, and protein translation. Collectively, these findings indicate a crucial role for microenvironment-mediated factors modulated by BMX in the escape from targeted therapy and have implications for the development of novel therapeutic interventions to restore sensitivity to gilteritinib.
  6. Leukemia. 2022 Jul 08.
      Recent studies highlighted the role of transcription factors in metabolic regulation during hematopoiesis and leukemia development. GFI1B is a transcriptional repressor that plays a critical role in hematopoiesis, and its expression is negatively related to the prognosis of acute myeloid leukemia (AML) patients. We earlier reported a change in the metabolic state of hematopoietic stem cells upon Gfi1b deletion. Here we explored the role of Gfi1b in metabolism reprogramming during hematopoiesis and leukemogenesis. We demonstrated that Gfi1b deletion remarkably activated mitochondrial respiration and altered energy metabolism dependence toward oxidative phosphorylation (OXPHOS). Mitochondrial substrate dependency was shifted from glucose to fatty acids upon Gfi1b deletion via upregulating fatty acid oxidation (FAO). On a molecular level, Gfi1b epigenetically regulated multiple FAO-related genes. Moreover, we observed that metabolic phenotypes evolved as cells progressed from preleukemia to leukemia, and the correlation between Gfi1b expression level and metabolic phenotype was affected by genetic variations in AML cells. FAO or OXPHOS inhibition significantly impeded leukemia progression of Gfi1b-KO MLL/AF9 cells. Finally, we showed that Gfi1b-deficient AML cells were more sensitive to metformin as well as drugs implicated in OXPHOS and FAO inhibition, opening new potential therapeutic strategies.
  7. Cancer Drug Resist. 2022 ;5(2): 344-367
      In acute myeloid leukemia (AML), a small cell population that contains stem cell features such as lack of differentiation, self-renewal potential, and drug resistance, can be identified. These so-called leukemic stem cells (LSCs) are thought to be responsible for relapse initiation after initial treatment leading to successful eradication of the bulk AML cell population. Since many studies have aimed to characterize and eliminate LSCs to prevent relapse and increase survival rates of patients, LSCs are one of the best characterized cancer stem cells. The specific elimination of LSCs, while sparing the healthy normal hematopoietic stem cells (HSCs), is one of the major challenges in the treatment of leukemia. This review focuses on several surface markers and intracellular transcription factors that can distinguish AML LSCs from HSCs and, therefore, specifically eliminate these stem cell-like leukemic cells. Moreover, previous and ongoing clinical trials of acute leukemia patients treated with therapies targeting these markers are discussed. In contrast to knowledge on LSCs in AML, insight into LSCs in acute lymphoid leukemia (ALL) is limited. This review therefore also addresses the latest insight into LSCs in ALL.
    Keywords:  Acute myeloid leukemia; acute lymphoid leukemia; leukemic stem cells; targeted therapy
  8. Blood Adv. 2022 Jul 06. pii: bloodadvances.2022007250. [Epub ahead of print]
      Genome-wide CRISPR screens have been extremely useful to identify therapeutic targets in diverse cancers, by defining genes that are essential for malignant growth. However, most CRISPR screens were performed in vitro and thus cannot identify genes that are essential for interactions with the microenvironment in vivo. Here, we report genome-wide CRISPR screens in two in vivo murine models of AML driven by the KMT2A/MLLT3 fusion or by the constitutive co-expression of Hoxa9 and Meis1. Secondary validation using a focused library identified 72 genes specifically essential for leukemic growth in vivo, including components of the MHC class I complex, Cd47, complement receptor Cr1l, and the beta-4-galactosylation pathway. Importantly, several of these in vivo-specific hits have prognostic effect or are inferred to be master regulators of protein activity in human AML cases. For instance, we identified Fermt3, a master regulator of integrin signaling, as an in vivo-specific dependency with high prognostic relevance. Overall, we demonstrate an experimental and computational pipeline for genome-wide functional screens in vivo in AML and provide a genome-wide resource of essential drivers of leukemic growth in vivo.
  9. Blood Adv. 2022 Jul 05. pii: bloodadvances.2022007544. [Epub ahead of print]
      Survival of patients with acute myeloid leukemia (AML) is inversely associated with age, but the impact of race on outcomes of adolescent and young adult (AYA, 18-39-year-old) patients is unknown. We compared survival of 89 Non-Hispanic Black AYA AML patients with survival of 566 Non-Hispanic White patients treated on frontline Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology protocols. Samples of 327 patients (50 Black and 277 White) were analyzed via targeted sequencing. Integrated genomic profiling was performed on select longitudinal samples. Black patients had worse outcomes, especially those aged 18-29 years, who had a higher early death rate (16% vs 3%, P=.002), lower complete remission rate (66% vs 83%, P=.01), and decreased overall survival (OS; 5-year rates: 22% vs 51%, P<.001) compared with White patients. Survival disparities persisted across cytogenetic groups: 18-29-year-old Black patients with non-core-binding-factor(CBF)-AML had worse OS than White patients (5-year rates: 12% vs 44%, P<.001), including patients with cytogenetically normal AML (13% vs 50%, P=0.003). Genetic features differed, including lower frequencies of normal karyotypes and NPM1 and bi-allelic CEBPA mutations, and higher frequencies of CBF rearrangements and ASXL1, BCOR, and KRAS mutations in Black patients. Integrated genomic analysis identified both known and novel somatic variants, and relative clonal stability at relapse. Reduced response rates to induction chemotherapy and leukemic clone persistence suggest a need for different treatment intensities and/or modalities in Black AYA patients with AML. Higher early death rates suggest a delay in diagnosis and treatment, calling for systematic changes to patient care.
  10. Am J Hematol. 2022 Jul 06.
      In most patients with chronic myeloid leukemia (CML) clonal cells can be kept under control by BCR::ABL1 tyrosine kinase inhibitors (TKI). However, overt resistance or intolerance against these TKI may occur. We identified the epigenetic reader BRD4 and its downstream-effector MYC as growth regulators and therapeutic targets in CML cells. BRD4 and MYC were found to be expressed in primary CML cells, CD34+ /CD38- leukemic stem cells (LSC), and in the CML cell lines KU812, K562, KCL22 and KCL22T315I . The BRD4-targeting drug JQ1 was found to suppress proliferation in KU812 cells and primary leukemic cells in the majority of patients with chronic phase CML. In the blast phase of CML, JQ1 was less effective. However, the BRD4 degrader dBET6 was found to block proliferation and/or survival of primary CML cells in all patients tested, including blast phase CML and CML cells exhibiting the T315I variant of BCR::ABL1. Moreover, dBET6 was found to block MYC expression and to synergize with BCR::ABL1 TKI in inhibiting the proliferation in the JQ1-resistant cell line K562. Furthermore, BRD4 degradation was found to overcome osteoblast-induced TKI resistance of CML LSC in a co-culture system and to block interferon-gamma-induced upregulation of the checkpoint antigen PD-L1 in LSC. Finally, dBET6 was found to suppress the in vitro survival of CML LSC and their engraftment in NSG mice. Together, targeting of BRD4 and MYC through BET degradation sensitizes CML cells against BCR::ABL1 TKI and is a potent approach to overcome multiple forms of drug resistance in CML LSC. This article is protected by copyright. All rights reserved.
  11. Sci Rep. 2022 Jul 05. 12(1): 11344
      Acute myeloid leukemia (AML) is characterized by arrested differentiation making differentiation therapy a promising treatment strategy. Recent success of inhibitors of mutated isocitrate dehydrogenase (IDH) invigorated interest in differentiation therapy of AML so that several new drugs have been proposed, including inhibitors of dihydroorotate dehydrogenase (DHODH), an enzyme in pyrimidine synthesis. Cytarabine, a backbone of standard AML therapy, is known to induce differentiation at low doses, but the mechanism is not completely elucidated. We have previously reported that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr) and brequinar, a DHODH inhibitor, induced differentiation of myeloid leukemia by activating the ataxia telangiectasia and Rad3-related (ATR)/checkpoint kinase 1 (Chk1) via pyrimidine depletion. In this study, using immunoblotting, flow cytometry analyses, pharmacologic inhibitors and genetic inactivation of Chk1 in myeloid leukemia cell lines, we show that low dose cytarabine induces differentiation by activating Chk1. In addition, cytarabine induces differentiation ex vivo in a subset of primary AML samples that are sensitive to AICAr and DHODH inhibitor. The results of our study suggest that leukemic cell differentiation stimulated by low doses of cytarabine depends on the activation of Chk1 and thus shares the same pathway as pyrimidine synthesis inhibitors.
  12. Nat Med. 2022 Jul 04.
      Clonal hematopoiesis of indeterminate potential (CHIP) increases rapidly in prevalence beyond age 60 and has been associated with increased risk for malignancy, heart disease and ischemic stroke. CHIP is driven by somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Because mutations in HSPCs often drive leukemia, we hypothesized that HSPC fitness substantially contributes to transformation from CHIP to leukemia. HSPC fitness is defined as the proliferative advantage over cells carrying no or only neutral mutations. If mutations in different genes lead to distinct fitness advantages, this could enable patient stratification. We quantified the fitness effects of mutations over 12 years in older age using longitudinal sequencing and developed a filtering method that considers individual mutational context alongside mutation co-occurrence to quantify the growth potential of variants within individuals. We found that gene-specific fitness differences can outweigh inter-individual variation and, therefore, could form the basis for personalized clinical management.
  13. J Immunother Cancer. 2022 Jul;pii: e004222. [Epub ahead of print]10(7):
      BACKGROUND: Acute myeloid leukemia (AML) remains a very difficult disease to cure due to the persistence of leukemic stem cells (LSCs), which are resistant to different lines of chemotherapy and are the basis of refractory/relapsed (R/R) disease in 80% of patients with AML not receiving allogeneic transplantation.METHODS: In this study, we showed that the interleukin-1 receptor accessory protein (IL-1RAP) protein is overexpressed on the cell surface of LSCs in all subtypes of AML and confirmed it as an interesting and promising target in AML compared with the most common potential AML targets, since it is not expressed by the normal hematopoietic stem cell. After establishing the proof of concept for the efficacy of chimeric antigen receptor (CAR) T-cells targeting IL-1RAP in chronic myeloid leukemia, we hypothesized that third-generation IL-1RAP CAR T-cells could eliminate AML LSCs, where the medical need is not covered.
    RESULTS: We first demonstrated that IL-1RAP CAR T-cells can be produced from AML T-cells at the time of diagnosis and at relapse. In vitro and in vivo, we showed the effectiveness of IL-1RAP CAR T-cells against AML cell lines expressing different levels of IL-1RAP and the cytotoxicity of autologous IL-1RAP CAR T-cells against primary cells from patients with AML at diagnosis or at relapse. In patient-derived relapsed AML xenograft models, we confirmed that IL-1RAP CAR T-cells are able to circulate in peripheral blood and to migrate in the bone marrow and spleen, are cytotoxic against primary AML cells and increased overall survival.
    CONCLUSION: In conclusion, our preclinical results suggest that IL-1RAP CAR T-based adoptive therapy could be a promising strategy in AML treatment and it warrants the clinical investigation of this CAR T-cell therapy.
    Keywords:  T-lymphocytes; cytotoxicity, immunologic; hematologic neoplasms; immunotherapy
  14. JCI Insight. 2022 Jul 05. pii: e159430. [Epub ahead of print]
      NLRP3 inflammasome and interferon stimulated gene (ISG) induction are key biological drivers of ineffective hematopoiesis and inflammation in Myelodysplastic Syndromes (MDS). Gene mutations involving messenger RNA splicing and epigenetic regulatory pathways induce inflammasome activation and myeloid lineage skewing in MDS through yet undefined mechanisms. Using immortalized murine hematopoietic stem and progenitor cells harboring these somatic gene mutations and primary MDS bone marrow specimens, we show accumulation of unresolved R-loops and micronuclei with concurrent activation of the cytosolic sensor, cGAS. cGAS-STING signaling caused interferon stimulated gene (ISG) induction, NLRP3 inflammasome activation, and maturation of the effector protease, caspase-1. Deregulation of RNA polymerase III drives cytosolic R-loop generation, which upon inhibition, extinguishes ISG and inflammasome response. Mechanistically, caspase-1 degrades the master erythroid transcription factor, GATA1, provoking anemia and myeloid lineage bias that is reversed by cGAS inhibition in vitro and in Tet2-/- hematopoietic stem and progenitor cell transplanted mice. Together, these data identity a novel mechanism by which functionally distinct mutations converge upon the cGAS-STING-NLRP3 axis in MDS directing ISG induction, pyroptosis and myeloid lineage skewing.
    Keywords:  Hematology; Leukemias; Oncology
  15. Cancer Discov. 2022 Jul 06. 12(7): 1617-1619
      Ellegast and colleagues show that monocytic acute myeloid leukemias (AML), enriched in inflammatory and immune gene sets, exploit a transcriptional repressor-namely, IRF2BP2-to mitigate their cell-intrinsic inflammatory output and ensure their maintenance. IRF2BP2 ablation results in heightened inflammatory signals that reach a set point that triggers apoptotic AML cell death in an NF-κB-IL1β-dependent manner. The study identifies IRF2BP2 as a cell-intrinsic vulnerability with potential therapeutic significance in monocytic AML. See related article by Ellegast et al., p. 1760 (6).
  16. Blood Cancer J. 2022 Jul 08. 12(7): 106
      Therapy-related myeloid neoplasms (t-MN) are aggressive leukemia that develops as a complication of prior exposure to DNA-damaging agents. Clonal cytopenia of undetermined significance (CCUS) is a precursor of de novo myeloid neoplasms. Characteristics of CCUS that develop following cytotoxic therapies (therapy-related clonal cytopenia, t-CC) and outcomes following t-CC have not been described. We identified 33 patients with t-CC and compared to a cohort of the WHO-defined t-MN (n = 309). t-CC had a distinct genetic and cytogenetic profile: pathogenic variants (PV) in TET2 and SRSF2 were enriched in t-CC, whereas TP53 PV was more common in t-MN. Ten (30%) t-CC patients developed a subsequent t-MN, with a cumulative incidence of 13%, 23%, and 50% at 6 months, 1, and 5 years, respectively. At t-MN progression, 44% of evaluable patients had identifiable clonal evolution. The median survival following t-CC was significantly superior compared all t-MN phenotype including t-MDS with <5% bone marrow blasts (124.5 vs. 16.3 months, P < 0.001) respectively. The presence of cytogenetic abnormality and the absence of variants in DNMT3A, TET2, or ASXL1 (DTA-genes) were associated with a higher likelihood of developing a subsequent t-MN and an inferior survival. We describe a putative precursor entity of t-MN with distinct features and outcomes.
  17. Blood Adv. 2022 Jun 24. pii: bloodadvances.2022007596. [Epub ahead of print]
      A common method to prevent graft versus host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT) from an HLA-mismatched unrelated donor (MMUD) is tacrolimus, methotrexate and anti-thymocyte globulin (ATG). The use of post-transplant cyclophosphamide (PTCy) demonstrated promise in a prospective trial for MMUD HCT. We compared one-year GVHD-free, relapse-free survival (GRFS) in 128 recipients of tacrolimus/methotrexate/ATG (ATG group, N = 46) with PTCy, mycophenolate mofetil, and tacrolimus or sirolimus (PTCy group, N = 82) based prophylaxis after MMUD HCT. Patients receiving HCT from a MMUD mismatched at ≥ 1 locus among HLA-A, -B, -C, and -DRB1 were included. The two groups were well matched for HCT indication, high-risk disease, and HCT comorbidity index, whereas more patients on PTCy received bone marrow (50% versus 26%, P = 0.01) and >1 locus HLA-mismatched grafts (30.5% vs. 2.2%, P = 0.001). The one-year GRFS was 16% (95% confidence interval: 8% - 31%) versus 54% (44% - 66%, P < 0.001) in the ATG and PTCy groups, respectively. The multivariable-adjusted hazard ratio (HR) for GRFS was 0.34 (0.21 - 0.55, P < 0.001) with the use of PTCy. The one-year overall survival (OS) in the ATG group was 45% (32% - 62%) versus 75% (66% - 85%) in the PTCy group (P < 0.001). Relapse incidence was similar. One-year non-relapse mortality was greater after ATG-based prophylaxis: (38%, 23% - 52%; versus 16%, 9% - 25%; P < 0.001). In summary, PTCy based prophylaxis resulted in superior GRFS and OS in recipients of MMUD.
  18. Cancer Discov. 2022 Jul 05. pii: cd.21.1671. [Epub ahead of print]
      Polycomb Repressive Complex 2 (PRC2) has oncogenic and tumor suppressor roles in cancer. There is clinical success of targeting this complex in PRC2-dependent cancers, but an unmet therapeutic need exists in PRC2-loss cancer. PRC2 inactivating mutations are a hallmark feature of high-grade malignant peripheral nerve sheath tumor (MPNST), an aggressive sarcoma with poor prognosis and no effective targeted therapy. Through RNAi screening in MPNST, we found that PRC2-inactivation increases sensitivity to genetic or small molecule inhibition of DNA methyltransferase 1 (DNMT1), which results in enhanced cytotoxicity and antitumor response. Mechanistically, PRC2 inactivation amplifies DNMT inhibitor (DNMTi) mediated expression of retrotransposons, subsequent viral mimicry response, and robust cell death in part through PKR-dependent double stranded-RNA (dsRNA) sensor. Collectively, our observations posit DNA methylation as a safeguard against anti-tumorigenic cell fate decisions in PRC2-loss cancer to promote cancer pathogenesis, which can be therapeutically exploited by DNMT1 targeted therapy.
  19. Cell Stem Cell. 2022 Jul 07. pii: S1934-5909(22)00253-3. [Epub ahead of print]29(7): 1119-1134.e7
      Hematopoietic stem cells (HSCs) adapt their metabolism to maintenance and proliferation; however, the mechanism remains incompletely understood. Here, we demonstrated that homeostatic HSCs exhibited high amino acid (AA) catabolism to reduce cellular AA levels, which activated the GCN2-eIF2α axis, a protein synthesis inhibitory checkpoint to restrain protein synthesis for maintenance. Furthermore, upon proliferation conditions, HSCs enhanced mitochondrial oxidative phosphorylation (OXPHOS) for higher energy production but decreased AA catabolism to accumulate cellular AAs, which inactivated the GCN2-eIF2α axis to increase protein synthesis and coupled with proteotoxic stress. Importantly, GCN2 deletion impaired HSC function in repopulation and regeneration. Mechanistically, GCN2 maintained proteostasis and inhibited Src-mediated AKT activation to repress mitochondrial OXPHOS in HSCs. Moreover, the glycolytic metabolite, NAD+ precursor nicotinamide riboside (NR), accelerated AA catabolism to activate GCN2 and sustain the long-term function of HSCs. Overall, our study uncovered direct links between metabolic alterations and translation control in HSCs during homeostasis and proliferation.
    Keywords:  GCN2; amino acid; hematopoietic stem cells; metabolism; nicotinamide riboside; oxidative phosphorylation; protein translation; proteostasis
  20. Blood. 2022 07 06. pii: blood.2022016985. [Epub ahead of print]
      Somatic mutations in UBA1 cause VEXAS (Vacuoles, E1 ubiquitin activating enzyme, X-linked, Autoinflammatory Somatic) syndrome, an adult-onset inflammatory disease with an overlap of hematologic manifestations. VEXAS syndrome is characterized by a high mortality rate and significant clinical heterogeneity. We sought to determine independent predictors of survival in VEXAS and to understand the mechanistic basis for these factors. We analyzed 83 patients with somatic pathogenic variants in UBA1 at p.Met41 (p.Met41Leu/Thr/Val), the start codon for translation of the cytoplasmic isoform of UBA1 (UBA1b). Patients with the p.Met41Val genotype were most likely to have an undifferentiated inflammatory syndrome. Multivariate analysis showed ear chondritis was associated with increased survival, while transfusion dependence and the p.Met41Val variant were independently associated with decreased survival. Using in vitro models and patient-derived cells, we demonstrate that p.Met41Val variant supports less UBA1b translation than either p.Met41Leu or p.Met41Thr, providing a molecular rationale for decreased survival. In addition, we show that these three canonical VEXAS variants produce more UBA1b than any of the six other possible single nucleotide variants within this codon. Finally, we report a patient, clinically diagnosed with VEXAS syndrome, with two novel mutations in UBA1 occurring in cis on the same allele. One mutation (c.121 A>T; p.Met41Leu) caused severely reduced translation of UBA1b in a reporter assay, but co-expression with the second mutation (c.119 G>C; p.Gly40Ala) rescued UBA1b levels to those of canonical mutations. We conclude that regulation of residual UBA1b translation is fundamental to the pathogenesis of VEXAS syndrome and contributes to disease prognosis.
  21. Nat Commun. 2022 Jul 04. 13(1): 3837
      Single-cell analysis methods are valuable tools; however, current approaches do not easily enable live cell retrieval. That is a particular issue when further study of cells that were eliminated during experimentation could provide critical information. We report a clonal molecular barcoding method, called SunCatcher, that enables longitudinal tracking and live cell functional analysis. From complex cell populations, we generate single cell-derived clonal populations, infect each with a unique molecular barcode, and retain stocks of individual barcoded clones (BCs). We develop quantitative PCR-based and next-generation sequencing methods that we employ to identify and quantify BCs in vitro and in vivo. We apply SunCatcher to various breast cancer cell lines and combine respective BCs to create versions of the original cell lines. While the heterogeneous BC pools reproduce their original parental cell line proliferation and tumor progression rates, individual BCs are phenotypically and functionally diverse. Early spontaneous metastases can also be identified and quantified. SunCatcher thus provides a rapid and sensitive approach for studying live single-cell clones and clonal evolution, and performing functional analyses.