bims-tremyl Biomed News
on Therapy resistance biology in myeloid leukemia
Issue of 2022–08–07
24 papers selected by
Paolo Gallipoli, Barts Cancer Institute, Queen Mary University of London
  1. Genomic landscape of patients with FLT3-mutated acute myeloid leukemia (AML) treated within the CALGB 10603/RATIFY trial.
  2. The impact of inflammation-induced tumor plasticity during myeloid transformation.
  3. Phase 3 Trial of Gilteritinib Plus Azacitidine Vs Azacitidine for Newly Diagnosed FLT3mut+ AML Ineligible for Intensive Chemotherapy.
  4. HDAC6 inhibition decreases leukemic stem cell expansion driven by Hedgehog hyperactivation by restoring primary ciliogenesis.
  5. FLT3 inhibitors for acute myeloid leukemia: successes, defeats, and emerging paradigms.
  6. Targeting the EIF2AK1 signaling pathway rescues red blood cell production in SF3B1-mutant myelodysplastic syndromes with ringed sideroblasts.
  7. Relative impact of residual cytogenetic abnormalities and flow cytometric measurable residual disease on outcome after allogeneic hematopoietic cell transplantation in adult acute myeloid leukemia.
  8. Second Malignancies among Older Patients with Classical Myeloproliferative Neoplasms Treated with Hydroxyurea.
  9. High-resolution structural variant profiling of myelodysplastic syndromes by optical genome mapping uncovers cryptic aberrations of prognostic and therapeutic significance.
  10. Mitochondrial Deep Dive into Hematopoietic Stem Cell Dormancy: Not Much Glycolysis but Plenty of Sluggish Lysosomes.
  11. Transcriptional variability accelerates preleukemia by cell diversification and perturbation of protein synthesis.
  12. BET bromodomain inhibition rescues PD-1-mediated T-cell exhaustion in acute myeloid leukemia.
  13. Single-cell analysis of acute lymphoblastic and lineage ambiguous leukemia - approaches and molecular insights.
  14. Fate mapping of hematopoietic stem cells reveals two pathways of native thrombopoiesis.
  15. Single cell genomics in AML: extending the frontiers of AML research.
  16. Clinical and biological impact of ATP-binding cassette transporter activity in adult acute myeloid leukemia.
  17. Accurate determination of CRISPR-mediated gene fitness in transplantable tumours.
  18. Induction of zinc finger protein RNF6 auto-ubiquitination for the treatment of myeloma and chronic myeloid leukemia.
  19. Synergistic prostaglandin E synthesis by myeloid and endothelial cells promotes fetal hematopoietic stem cell expansion in vertebrates.
  20. TP53-dependent toxicity of CRISPR/Cas9 cuts is differential across genomic loci and can confound genetic screening.
  21. Transcriptome variation in human tissues revealed by long-read sequencing.
  22. Droplet digital PCR for the detection of second-generation tyrosine kinase inhibitor-resistant BCR::ABL1 kinase domain mutations in chronic myeloid leukemia.
  23. Turning the clock forward: Inflammation accelerates the aging of hematopoietic stem cells.
  24. Dual-antigen targeted off-the-shelf NK cells show durable response and prevent antigen escape in lymphoma and leukemia.
  1. Leukemia. 2022 Aug 03.
    Genomic landscape of patients with FLT3-mutated acute myeloid leukemia (AML) treated within the CALGB 10603/RATIFY trial.
    Nikolaus Jahn, Ekaterina Jahn, Maral Saadati, Lars Bullinger, Richard A Larson, Tiziana Ottone, Sergio Amadori, Thomas W Prior, Joseph M Brandwein, Frederick R Appelbaum, Bruno C Medeiros, Martin S Tallman, Gerhard Ehninger, Michael Heuser, Arnold Ganser, Celine Pallaud, Insa Gathmann, Julia Krzykalla, Axel Benner, Clara D Bloomfield, Christian Thiede, Richard M Stone, Hartmut Döhner, Konstanze Döhner.
      The aim of this study was to characterize the mutational landscape of patients with FLT3-mutated acute myeloid leukemia (AML) treated within the randomized CALGB 10603/RATIFY trial evaluating intensive chemotherapy plus the multi-kinase inhibitor midostaurin versus placebo. We performed sequencing of 262 genes in 475 patients: mutations occurring concurrently with the FLT3-mutation were most frequent in NPM1 (61%), DNMT3A (39%), WT1 (21%), TET2 (12%), NRAS (11%), RUNX1 (11%), PTPN11 (10%), and ASXL1 (8%) genes. To assess effects of clinical and genetic features and their possible interactions, we fitted random survival forests and interpreted the resulting variable importance. Highest prognostic impact was found for WT1 and NPM1 mutations, followed by white blood cell count, FLT3 mutation type (internal tandem duplications vs. tyrosine kinase domain mutations), treatment (midostaurin vs. placebo), ASXL1 mutation, and ECOG performance status. When evaluating two-fold variable combinations the most striking effects were found for WT1:NPM1 (with NPM1 mutation abrogating the negative effect of WT1 mutation), and for WT1:treatment (with midostaurin exerting a beneficial effect in WT1-mutated AML). This targeted gene sequencing study provides important, novel insights into the genomic background of FLT3-mutated AML including the prognostic impact of co-mutations, specific gene-gene interactions, and possible treatment effects of midostaurin.
    DOI:  https://doi.org/10.1038/s41375-022-01650-w
  2. Cancer Discov. 2022 Aug 04. pii: CD-21-1146. [Epub ahead of print]
    The impact of inflammation-induced tumor plasticity during myeloid transformation.
    Anna Yeaton, Geraldine Cayanan, Sanam Loghavi, Igor Dolgalev, Emmett M Leddin, Christian E Loo, Hedieh Torabifard, Deedra Nicolet, Jingjing Wang, Kate Corrigan, Varvara Paraskevopoulou, Daniel T Starczynowski, Eric Wang, Omar Abdel-Wahab, Aaron D Viny, Richard M Stone, John C Byrd, Olga A Guryanova, Rahul M Kohli, G Andres Cisneros, Aristotelis Tsirigos, Ann-Kathrin Eisfeld, Iannis Aifantis, Maria Guillamot.
      Clonal hematopoiesis (CH) is an aging-associated condition characterized by the clonal outgrowth of mutated pre-leukemic cells. Individuals with CH are at an increased risk of developing hematopoietic malignancies. Here, we describe a novel animal model carrying a recurrent TET2 missense mutation, frequently found in CH and leukemic patients. In a fashion similar to CH, animals show signs of disease late in life when they develop a wide range of myeloid neoplasms, including acute myeloid leukemia (AML). Using single cell transcriptomic profiling of the bone marrow, we show that disease progression in aged animals correlates with an enhanced inflammatory response and the emergence of an aberrant inflammatory monocytic cell population. The gene signature characteristic of this inflammatory population is associated to poor prognosis in AML patients. Our study illustrates an example of collaboration between a genetic lesion found in CH and inflammation, leading to transformation and the establishment of blood neoplasms.
    DOI:  https://doi.org/10.1158/2159-8290.CD-21-1146
  3. Blood. 2022 Aug 02. pii: blood.2021014586. [Epub ahead of print]
    Phase 3 Trial of Gilteritinib Plus Azacitidine Vs Azacitidine for Newly Diagnosed FLT3mut+ AML Ineligible for Intensive Chemotherapy.
    Eunice S Wang, Pau Montesinos, Mark D Minden, Je-Hwan Lee, Michael Heuser, Tomoki Naoe, Wen-Chien Chou, Kamel Laribi, Jordi Esteve, Jessica K Altman, Violaine Havelange, Anne-Marie Watson, Carlo B Gambacorti-Passerini, Elzbieta Natasza Patkowska, Shufang Liu, Ruishan Wu, Nisha Philipose, Jason E Hill, Stanley C Gill, Elizabeth Shima Rich, Ramon Tiu.
      Treatment results for patients with newly diagnosed FMS-like tyrosine kinase 3 (FLT3)-mutated (FLT3mut+) acute myeloid leukemia (AML) ineligible for intensive chemotherapy are disappointing. This multicenter, open-label, phase 3 trial randomized (2:1) untreated adults with FLT3mut+ AML ineligible for intensive induction chemotherapy to gilteritinib (120 mg/day orally) and azacitidine (GIL+AZA) or azacitidine (AZA) alone. The primary endpoint was overall survival (OS). At the interim analysis (26 August 2020), 123 patients were randomized (GIL+AZA, n=74; AZA, n=49). Subsequent AML therapy, including FLT3 inhibitors, was received by 20.3% (GIL+AZA) and 44.9% (AZA) of patients. Median OS was 9.82 (GIL+AZA) and 8.87 (AZA) months (HR 0.916 [95% CI: 0.529, 1.585]; P=0.753). The study was closed based on the protocol-specified boundary for futility. Median event-free survival (EFS) was 0.03 months in both arms. EFS defined using composite CR (CRc) was 4.53 (GIL+AZA) and 0.03 (AZA) months (HR 0.686 [95% CI: 0.433, 1.087]; P=0.156). CRc rates were 58.1% (GIL+AZA) and 26.5% (AZA; difference 31.4% [95% CI: 13.1, 49.7]; P<0.001). Adverse event (AE) rates were similar for GIL+AZA (100%) and AZA (95.7%) and grade ≥3 AEs were 95.9% and 89.4%, respectively. Common AEs with GIL+AZA included pyrexia (47.9%) and diarrhea (38.4%). Gilteritinib steady state trough concentrations were no different between GIL+AZA and gilteritinib. GIL+AZA resulted in significantly higher CRc rates although similar OS versus AZA. Results support the safety/tolerability and clinical activity of upfront therapy with GIL+AZA in older/unfit patients with FLT3mut+ AML. Clinical Trial # NCT02752035.
    DOI:  https://doi.org/10.1182/blood.2021014586
  4. Pharmacol Res. 2022 Jul 30. pii: S1043-6618(22)00323-1. [Epub ahead of print] 106378
    HDAC6 inhibition decreases leukemic stem cell expansion driven by Hedgehog hyperactivation by restoring primary ciliogenesis.
    Alex Pezzotta, Ilaria Gentile, Donatella Genovese, Maria Grazia Totaro, Cristina Battaglia, Anskar Yu-Hung Leung, Monica Fumagalli, Matteo Parma, Gianni Cazzaniga, Grazia Fazio, Myriam Alcalay, Anna Marozzi, Anna Pistocchi.
      Aberrant activation of the Hh pathway promotes cell proliferation and multi-drug resistance (MDR) in several cancers, including Acute Myeloid Leukemia (AML). Notably, only one Hh inhibitor, glasdegib, has been approved for AML treatment, and most patients eventually relapse, highlighing the urgent need ti discover new therapeutic targets. Hh signal is transduced through the membrane of the primary cilium, a structure expressed by non-proliferating mammalian cells, whose stabilization depends on the activity of HDAC6. Here we describe a positive correlation between Hh, HDAC6, and MDR genes in a cohort of adult AML patients, human leukemic cell lines, and a zebrafish model of Hh overexpression. The hyper-activation of Hh or HDAC6 in zebrafish drove the increased proliferation of hematopoietic stem and progenitor cells (HSPCs). Interestingly, this phenotype was rescued by inhibition of HDAC6 but not of Hh. Also, in human leukemic cell lines, a reduction in vitality was obtained through HDAC6, but not Hh inhibition. Our data showed the presence of a cross-talk between Hh and HDAC6 mediated by stabilization of the primary cilium, which we detect for the first time in zebrafish HSPCs. Inhibition of HDAC6 activity alone or in combination therapy with the chemotherapeutic agent cytarabine, efficiently rescued the hematopoietic phenotype. Our results open the possibility to introduce HDAC6 as therapeutic target to reduce proliferation of leukemic blasts in AML patients.
    Keywords:  AML; Dymetil sulfoxide D8418 (Sigma); Glasdegib PZ0303 (Sigma); HDAC6; Hedgehog; Roscovitine R7772 (Sigma); TubastatinA hydrochloride SML0044 (Sigma); cyclopamine hydrate C4116 (Sigma); cytarabine PHR1787 (Sigma); primary cilium; zebrafish
    DOI:  https://doi.org/10.1016/j.phrs.2022.106378
  5. RSC Med Chem. 2022 Jul 20. 13(7): 798-816
    FLT3 inhibitors for acute myeloid leukemia: successes, defeats, and emerging paradigms.
    Baku Acharya, Debasmita Saha, Daniel Armstrong, Naga Rajiv Lakkaniga, Brendan Frett.
      FLT3 mutations are one of the most common genetic aberrations found in nearly 30% of acute myeloid leukemias (AML). The mutations are associated with poor prognosis despite advances in the understanding of the biological mechanisms of AML. Numerous small molecule FLT3 inhibitors have been developed in an effort to combat AML. Even with the development of these inhibitors, the five-year overall survival for newly diagnosed AML is less than 30%. In 2017, midostaurin received FDA approval to treat AML, which was the first approved FLT3 inhibitor in the U.S. and Europe. Following, gilteritinib received FDA approval in 2018 and in 2019 quizartinib received approval in Japan. This review parallels these clinical success stories along with other pre-clinical and clinical investigations of FLT3 inhibitors.
    DOI:  https://doi.org/10.1039/d2md00067a
  6. Blood Cancer Discov. 2022 Aug 04. pii: BCD-21-0220. [Epub ahead of print]
    Targeting the EIF2AK1 signaling pathway rescues red blood cell production in SF3B1-mutant myelodysplastic syndromes with ringed sideroblasts.
    Vera Adema, Feiyang Ma, Rashmi Kanagal-Shamanna, Natthakan Thongon, Guillermo Montalban-Bravo, Hui Yang, Scott A Peslak, Feng Wang, Pamela Acha, Francesc Sole, Pamela Lockyer, Margherita Cassari, Jaroslaw P Maciejewski, Valeria Visconte, Irene Ganan-Gomez, Yuanbin Song, Carlos Bueso-Ramos, Matteo Pellegrini, Tuyet M Tan, Rafael Bejar, Jennifer S Carew, Stephanie Halene, Valeria Santini, Gheath Al-Atrash, Karen Clise-Dwyer, Guillermo Garcia-Manero, Gerd A Blobel, Simona Colla.
      SF3B1 mutations, which occur in 20% of patients with myelodysplastic syndromes (MDS), are the hallmarks of a specific MDS subtype, MDS with ringed sideroblasts (MDS-RS), which is characterized by the accumulation of erythroid precursors in the bone marrow and primarily affects the elderly population. Here, using single-cell technologies and functional validation studies of primary SF3B1-mutant MDS-RS samples, we show that SF3B1 mutations lead to the activation of the EIF2AK1 pathway in response to heme deficiency and that targeting this pathway rescues aberrant erythroid differentiation and enables the red blood cell maturation of MDS-RS erythroblasts. These data support the development of EIF2AK1 inhibitors to overcome transfusion dependency in patients with SF3B1-mutant MDS-RS with impaired red blood cell production.
    DOI:  https://doi.org/10.1158/2643-3230.BCD-21-0220
  7. Haematologica. 2022 Aug 04.
    Relative impact of residual cytogenetic abnormalities and flow cytometric measurable residual disease on outcome after allogeneic hematopoietic cell transplantation in adult acute myeloid leukemia.
    Corentin Orvain, Jacob A Wilson, Min Fang, Brenda M Sandmaier, Eduardo Rodríguez-Arbolí, Brent L Wood, Megan Othus, Frederick R Appelbaum, Roland B Walter.
      Measurable residual disease (MRD) before hematopoietic cell transplantation (HCT) is an independent established prognostic factor in patients with acute myeloid leukemia (AML). Several methods exist to evaluate the presence of residual leukemia cells, but how these are used best in combination is unclear. To examine how residual cytogenetic abnormalities and MRD testing by multiparameter flow cytometry (MFC) may refine risk assessment before HCT, we analyzed 506 adults with cytogenetically abnormal AML who underwent both routine karyotyping and MFC MRD testing before receiving a first allograft while in morphologic remission. Testing for residual cytogenetic abnormalities and MFC MRD identified four groups of patients with differential relapse-free survival (RFS, hazard ratio [HR]=1.63 for Cytoabnormal/MFCnegative [P=0.01, n=63], HR=3.24 for Cytonormal/MFCpositive [P.
    DOI:  https://doi.org/10.3324/haematol.2022.281585
  8. Blood Adv. 2022 Aug 02. pii: bloodadvances.2022008259. [Epub ahead of print]
    Second Malignancies among Older Patients with Classical Myeloproliferative Neoplasms Treated with Hydroxyurea.
    Rong Wang, Rory Michael Shallis, Jessica M Stempel, Scott F Huntington, Amer M Zeidan, Steven D Gore, Xiaomei Ma, Nikolai A Podoltsev.
      Patients with classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPN), including polycythemia vera (PV), essential thrombocythemia (ET), primary and secondary myelofibrosis (MF), are known to have an increased risk of second malignancies (SM). Hydroxyurea (HU) is a guideline-recommended cytoreductive therapy for high-risk MPN patients. Controversy exists as to whether HU use is associated with a higher risk of SM including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We conducted a retrospective cohort study of older patients diagnosed with MPN (age ≥66 years) in 2010-2017 and included in the Surveillance, Epidemiology, and End Results-Medicare linked database. Multivariable competing risk analyses adjusting for patient characteristics were utilized to assess the impact of HU on the development of SM. We identified 4023 patients (1688 with PV, 1976 with ET, and 359 with MF) with a median age of 77 (interquartile range [IQR]: 71-83) years at the time of MPN diagnosis. After a median follow-up of 3.25 (IQR: 2.10-5.00) years, 489 patients developed a SM (346 solid, 73 lymphoid, and 70 myeloid malignancies). The cumulative incidence probability of SM was 19.88% (95% confidence interval [CI] 17.16-22.75%) among 2683 HU users and 22.31% (95% CI: 17.51-27.47%) among 1340 non-users, respectively (Gray's test p<0.01). We did not identify significant differences in the incidence of solid or hematologic SM, including AML/MDS (HR=1.33, 95% CI: 0.77-2.29; p=0.30), between HU users and non-users. Our results suggest that the use of HU does not increase the risk of SM in older MPN patients.
    DOI:  https://doi.org/10.1182/bloodadvances.2022008259
  9. Leukemia. 2022 Aug 01.
    High-resolution structural variant profiling of myelodysplastic syndromes by optical genome mapping uncovers cryptic aberrations of prognostic and therapeutic significance.
    Hui Yang, Guillermo Garcia-Manero, Koji Sasaki, Guillermo Montalban-Bravo, Zhenya Tang, Yue Wei, Tapan Kadia, Kelly Chien, Diana Rush, Ha Nguyen, Awdesh Kalia, Manjunath Nimmakayalu, Carlos Bueso-Ramos, Hagop Kantarjian, L Jeffrey Medeiros, Rajyalakshmi Luthra, Rashmi Kanagal-Shamanna.
      Chromosome banding analysis (CBA) remains the standard-of-care for structural variant (SV) assessment in MDS. Optical genome mapping (OGM) is a novel, non-sequencing-based technique for high-resolution genome-wide SV profiling (SVP). We explored the clinical value of SVP by OGM in 101 consecutive, newly diagnosed MDS patients from a single-center, who underwent standard-of-care cytogenetic and targeted NGS studies. OGM detected 383 clinically significant, recurrent and novel SVs. Of these, 224 (51%) SVs, seen across 34% of patients, were cryptic by CBA (included rearrangements involving MECOM, NUP98::PRRX2, KMT2A partial tandem duplications among others). SVP decreased the proportion of normal karyotype by 16%, identified complex genomes (17%), chromothripsis (6%) and generated informative results in both patients with insufficient metaphases. Precise gene/exon-level mapping allowed assessment of clinically relevant biomarkers (TP53 allele status, KMT2A-PTD) without additional testing. SV data was complementary to NGS. When applied in retrospect, OGM results changed the comprehensive cytogenetic scoring system (CCSS) and R-IPSS risk-groups in 21% and 17% patients respectively with an improved prediction of prognosis. By multivariate analysis, CCSS by OGM only (not CBA), TP53 mutation and BM blasts independently predicted survival. This is the first and largest study reporting the value of combined SVP and NGS for MDS prognostication.
    DOI:  https://doi.org/10.1038/s41375-022-01652-8
  10. Exp Hematol. 2022 Jul 28. pii: S0301-472X(22)00577-X. [Epub ahead of print]
    Mitochondrial Deep Dive into Hematopoietic Stem Cell Dormancy: Not Much Glycolysis but Plenty of Sluggish Lysosomes.
    Jiajing Qiu, Saghi Ghaffari.
      The discovery of hematopoietic stem cells (HSCs) heterogeneity have had major implications for investigations of hematopoietic stem cell disorders, clonal hematopoiesis and HSC aging. More recent studies of the heterogeneity of HSC organelles have begun to provide additional insights into the HSC behavior with far reaching ramifications for mechanistic understanding of aging of HSCs and stem cell-derived diseases. Mitochondrial heterogeneity has been explored to expose HSC subsets with distinct properties and functions. Here we review some of the recent advances in these lines of studies that challenged the classical view of glycolysis in HSCs and led to the identification of lysosomes as dynamic pivotal switch in controlling HSC quiescence vs. activation beyond their function in autophagy.
    DOI:  https://doi.org/10.1016/j.exphem.2022.07.299
  11. Sci Adv. 2022 Aug 05. 8(31): eabn4886
    Transcriptional variability accelerates preleukemia by cell diversification and perturbation of protein synthesis.
    Shikha Gupta, Oliver M Dovey, Ana Filipa Domingues, Oliwia W Cyran, Caitlin M Cash, George Giotopoulos, Justyna Rak, Jonathan Cooper, Malgorzata Gozdecka, Liza Dijkhuis, Ryan J Asby, Noor Al-Jabery, Victor Hernandez-Hernandez, Sudhakaran Prabakaran, Brian J Huntly, George S Vassiliou, Cristina Pina.
      Transcriptional variability facilitates stochastic cell diversification and can in turn underpin adaptation to stress or injury. We hypothesize that it may analogously facilitate progression of premalignancy to cancer. To investigate this, we initiated preleukemia in mouse cells with enhanced transcriptional variability due to conditional disruption of the histone lysine acetyltransferase gene Kat2a. By combining single-cell RNA sequencing of preleukemia with functional analysis of transformation, we show that Kat2a loss results in global variegation of cell identity and accumulation of preleukemic cells. Leukemia progression is subsequently facilitated by destabilization of ribosome biogenesis and protein synthesis, which confer a transient transformation advantage. The contribution of transcriptional variability to early cancer evolution reflects a generic role in promoting cell fate transitions, which, in the case of well-adapted malignancies, contrastingly differentiates and depletes cancer stem cells. That is, transcriptional variability confers forward momentum to cell fate systems, with differential multistage impact throughout cancer evolution.
    DOI:  https://doi.org/10.1126/sciadv.abn4886
  12. Cell Death Dis. 2022 Aug 02. 13(8): 671
    BET bromodomain inhibition rescues PD-1-mediated T-cell exhaustion in acute myeloid leukemia.
    Mengjun Zhong, Rili Gao, Ruocong Zhao, Youxue Huang, Cunte Chen, Kehan Li, Xibao Yu, Dingrui Nie, Zheng Chen, Xin Liu, Zhuandi Liu, Shaohua Chen, Yuhong Lu, Zhi Yu, Liang Wang, Peng Li, Chengwu Zeng, Yangqiu Li.
      Sustained expression of programmed cell death receptor-1 (PD-1) is correlated with the exhaustion of T cells, and blockade of the PD-1 pathway is an effective immunotherapeutic strategy for treating various cancers. However, response rates are limited, and many patients do not achieve durable responses. Thus, it is important to seek additional strategies that can improve anticancer immunity. Here, we report that the bromodomain and extraterminal domain (BET) inhibitor JQ1 inhibits PD-1 expression in Jurkat T cells, primary T cells, and T-cell exhaustion models. Furthermore, JQ1 dramatically impaired the expression of PD-1 and T-cell immunoglobulin mucin-domain-containing-3 (Tim-3) and promoted the secretion of cytokines in T cells from patients with acute myeloid leukemia (AML). In line with that, BET inhibitor-treated CD19-CAR T and CD123-CAR T cells have enhanced anti-leukemia potency and resistant to exhaustion. Mechanistically, BRD4 binds to the NFAT2 and PDCD1 (encoding PD-1) promoters, and NFAT2 binds to the PDCD1 and HAVCR2 (encoding Tim-3) promoters. JQ1-treated T cells showed downregulated NFAT2, PD-1, and Tim-3 expression. In addition, BET inhibitor suppressed programmed death-ligand 1 (PD-L1) expression and cell growth in AML cell lines and in primary AML cells. We also demonstrated that JQ1 treatment led to inhibition of leukemia progression, reduced T-cell PD-1/Tim-3 expression, and prolonged survival in MLL-AF9 AML mouse model and Nalm6 (B-cell acute lymphoblastic leukemia cell)-bearing mouse leukemia model. Taken together, BET inhibition improved anti-leukemia immunity by regulating PD-1/PD-L1 expression, and also directly suppressed AML cells, which provides novel insights on the multiple effects of BET inhibition for cancer therapy.
    DOI:  https://doi.org/10.1038/s41419-022-05123-x
  13. Blood. 2022 Aug 04. pii: blood.2022016954. [Epub ahead of print]
    Single-cell analysis of acute lymphoblastic and lineage ambiguous leukemia - approaches and molecular insights.
    Ilaria Iacobucci, Matthew T Witkowski, Charles G Mullighan.
      Despite recent progress in identifying the genetic drivers of acute lymphoblastic leukemia (ALL), prognosis remains poor for those individuals who experience disease recurrence. Moreover, acute leukemias of ambiguous lineage (ALAL) lack a biologically-informed framework to guide classification and therapy. These needs have driven the adoption of multiple complementary single-cell sequencing approaches to explore key issues in the biology of these leukemias, including cell of origin, developmental hierarchy and ontogeny, and molecular heterogeneity driving pathogenesis, progression, and therapeutic responsiveness in ALL and related leukemias. There are multiple single cell techniques to profile a specific modality, including RNA, DNA, chromatin accessibility and methylation, and an expanding range of approaches to simultaneously analyze more than one modality. Single-cell sequencing approaches have also enabled characterization of cell-intrinsic and -extrinsic features of ALL biology. In this review we describe these approaches and highlight the extensive heterogeneity that underpins ALL gene expression, cellular differentiation and clonal architecture throughout disease pathogenesis and treatment resistance. In addition, we discuss the importance of dynamic interactions occurring between leukemia cells and the non-leukemia microenvironment. We discuss potential opportunities and limitations of single-cell sequencing for the study of ALL biology and treatment responsiveness.
    DOI:  https://doi.org/10.1182/blood.2022016954
  14. Nat Commun. 2022 Aug 03. 13(1): 4504
    Fate mapping of hematopoietic stem cells reveals two pathways of native thrombopoiesis.
    Mina N F Morcos, Congxin Li, Clara M Munz, Alessandro Greco, Nicole Dressel, Susanne Reinhardt, Katrin Sameith, Andreas Dahl, Nils B Becker, Axel Roers, Thomas Höfer, Alexander Gerbaulet.
      Hematopoietic stem cells (HSCs) produce highly diverse cell lineages. Here, we chart native lineage pathways emanating from HSCs and define their physiological regulation by computationally integrating experimental approaches for fate mapping, mitotic tracking, and single-cell RNA sequencing. We find that lineages begin to split when cells leave the tip HSC population, marked by high Sca-1 and CD201 expression. Downstream, HSCs either retain high Sca-1 expression and the ability to generate lymphocytes, or irreversibly reduce Sca-1 level and enter into erythro-myelopoiesis or thrombopoiesis. Thrombopoiesis is the sum of two pathways that make comparable contributions in steady state, a long route via multipotent progenitors and CD48hi megakaryocyte progenitors (MkPs), and a short route from HSCs to developmentally distinct CD48-/lo MkPs. Enhanced thrombopoietin signaling differentially accelerates the short pathway, enabling a rapid response to increasing demand. In sum, we provide a blueprint for mapping physiological differentiation fluxes from HSCs and decipher two functionally distinct pathways of native thrombopoiesis.
    DOI:  https://doi.org/10.1038/s41467-022-31914-z
  15. Blood. 2022 Aug 04. pii: blood.2021014670. [Epub ahead of print]
    Single cell genomics in AML: extending the frontiers of AML research.
    Asiri Ediriwickrema, Andrew J Gentles, Ravindra Majeti.
      The era of genomic medicine has allowed AML researchers to improve disease characterization, optimize risk stratification systems, and develop new treatments. While there has been significant progress, AML remains a lethal cancer due to its remarkably complex and plastic cellular architecture. This degree of heterogeneity continues to pose a major challenge as it limits the ability to identify and therefore eradicate the cells responsible for leukemogenesis and treatment failures. In recent years, the field of single cell genomics has led to unprecedented strides in the ability to characterize cellular heterogeneity and holds promise for the study of AML. In this review, we will highlight advancements in single cell technologies, outline important shortcomings in our understanding of AML biology and clinical management, and discuss how single cell genomics can not only address these shortcomings, but also provide unique opportunities in basic and translational AML research.
    DOI:  https://doi.org/10.1182/blood.2021014670
  16. Haematologica. 2022 Aug 04.
    Clinical and biological impact of ATP-binding cassette transporter activity in adult acute myeloid leukemia.
    Elise Sourdeau, Ludovic Suner, Mara Memoli, Alexis Genthon, Frédéric Feger, Lou Soret, Nasséra Abermil, Laurence Heuberger, Chrystele Bilhou-Nabera, Hélène Guermouche, Fabrizia Favale, Simona Lapusan, Michael Chaquin, Claire Hirschauer, Mohamad Mohty, Ollivier Legrand, François Delhommeau, Pierre Hirsch.
      Chemotherapy resistance is the main cause of treatment failure in acute myeloid leukemia (AML) and has been related to ATP-binding cassette (ABC) transporter activity. However, the links between ABC activity, immunophenotype, and molecular AML parameters have been poorly evaluated. Moreover, the prognostic value of ABC activity, when compared to new molecular markers, is unknown. Here we investigated the links between ABC activity, as evaluated by JC-1 +/- cyclosporine A (CsA) assay, and immunophenotypic, cytogenetic, molecular, and targeted next generation sequencing features in 361 AML patients. High ABC activity was found in 164 patients and was significantly associated with less proliferating disease, an immature immunophenotype (expression of CD34, HLA-DR, CD117, CD13), and gene mutations defining AML as belonging to secondary-type ontogenic groups. Low ABC activity was associated with more mature myeloid differentiation (CD34-, cyMPO+, CD15+, CD33+) or monocytic commitment (CD64+, CD4+weak, CD14+), with NPM1 mutations, KMT2A rearrangements, and core-binding factor gene fusions, hallmarks of the de novotype AML ontogeny. ABC activity was one of the major factors we identified using a random forest model for early prediction of AML ontogeny. In the 230 patients evaluated at diagnosis and intensively treated, high ABC activity was a predictive factor for primary resistance, and in multivariate analysis including full molecular data, an independent factor for event-freesurvival (p=0.0370). JC-1 +/- CsA assay could be used at diagnosis to predict AML ontogeny and to complete prognosis evaluation in addition to new molecular markers.
    DOI:  https://doi.org/10.3324/haematol.2022.280676
  17. Nat Commun. 2022 Aug 04. 13(1): 4534
    Accurate determination of CRISPR-mediated gene fitness in transplantable tumours.
    Peter Eirew, Ciara O'Flanagan, Jerome Ting, Sohrab Salehi, Jazmine Brimhall, Beixi Wang, Justina Biele, Teresa Algara, So Ra Lee, Corey Hoang, Damian Yap, Steven McKinney, Cherie Bates, Esther Kong, Daniel Lai, Sean Beatty, Mirela Andronescu, Elena Zaikova, Tyler Funnell, Nicholas Ceglia, Stephen Chia, Karen Gelmon, Colin Mar, Sohrab Shah, Andrew Roth, Alexandre Bouchard-Côté, Samuel Aparicio.
      Assessing tumour gene fitness in physiologically-relevant model systems is challenging due to biological features of in vivo tumour regeneration, including extreme variations in single cell lineage progeny. Here we develop a reproducible, quantitative approach to pooled genetic perturbation in patient-derived xenografts (PDXs), by encoding single cell output from transplanted CRISPR-transduced cells in combination with a Bayesian hierarchical model. We apply this to 181 PDX transplants from 21 breast cancer patients. We show that uncertainty in fitness estimates depends critically on the number of transplant cell clones and the variability in clone sizes. We use a pathway-directed allelic series to characterize Notch signaling, and quantify TP53 / MDM2 drug-gene conditional fitness in outlier patients. We show that fitness outlier identification can be mirrored by pharmacological perturbation. Overall, we demonstrate that the gene fitness landscape in breast PDXs is dominated by inter-patient differences.
    DOI:  https://doi.org/10.1038/s41467-022-31830-2
  18. J Biol Chem. 2022 Aug 01. pii: S0021-9258(22)00756-6. [Epub ahead of print] 102314
    Induction of zinc finger protein RNF6 auto-ubiquitination for the treatment of myeloma and chronic myeloid leukemia.
    Haixia Zhuang, Ying Ren, Chenyu Mao, Yueya Zhong, Zubin Zhang, Biyin Cao, Yuming Zhang, Jinqi Huang, Guoqiang Xu, Zhenqian Huang, Yujia Xu, Xinliang Mao.
      The zinc finger ubiquitin ligase RNF6 has been proposed as a potential therapeutic target in several cancers, but understanding its molecular mechanism of degradation has been elusive. In the present study, we find that RNF6 is degraded via auto-ubiquitination in a manner dependent on its Really Interesting New Gene (RING) domain. We determine that when the RING domain is deleted (ΔRING) or the core cysteine residues in the zinc finger are mutated (C632S/C635S), the wild-type protein, but not the ΔRING or mutant RNF6 protein, undergoes polyubiquitination. We also identify USP7 as a deubiquitinase of RNF6 by tandem mass spectrometry. We show that USP7 interacts with RNF6 and abolishes its K48-linked polyubiquitination, thereby preventing its degradation. In contrast, we found a USP7-specific inhibitor promotes RNF6 polyubiquitination, degradation, and cell death. Furthermore, we demonstrate anti-leukemic drug Nilotinib and anti-myeloma drug Panobinostat (LBH589) induce RNF6 K48-linked polyubiquitination and degradation in both multiple myeloma (MM) and leukemia cells. In agreement with our hypothesis on the mode of RNF6 degradation, we show these drugs promote RNF6 auto-ubiquitination in an in vitro ubiquitination system without other E3 ligases. Consistently, re-expression of RNF6 ablates drug-induced MM and leukemia cell apoptosis. Therefore, our results reveal that RNF6 is a RING E3 ligase that undergoes auto-ubiquitination, which could be abolished by USP7 and induced by anti-cancer drugs. We propose chemical induction of RNF6 auto-ubiquitination and degradation could be a novel strategy for the treatment of hematological malignancies including MM and leukemia.
    Keywords:  RNF6; USP7; auto-ubiquitination; leukemia; myeloma
    DOI:  https://doi.org/10.1016/j.jbc.2022.102314
  19. EMBO J. 2022 Aug 04. e108536
    Synergistic prostaglandin E synthesis by myeloid and endothelial cells promotes fetal hematopoietic stem cell expansion in vertebrates.
    Pietro Cacialli, Marie-Pierre Mailhe, Ingrid Wagner, Doron Merkler, Rachel Golub, Julien Y Bertrand.
      During development, hematopoietic stem cells (HSCs) are produced from the hemogenic endothelium and will expand in a transient hematopoietic niche. Prostaglandin E2 (PGE2) is essential during vertebrate development and HSC specification, but its precise source in the embryo remains elusive. Here, we show that in the zebrafish embryo, PGE2 synthesis genes are expressed by distinct stromal cell populations, myeloid (neutrophils, macrophages), and endothelial cells of the caudal hematopoietic tissue. Ablation of myeloid cells, which produce the PGE2 precursor prostaglandin H2 (PGH2), results in loss of HSCs in the caudal hematopoietic tissue, which could be rescued by exogeneous PGE2 or PGH2 supplementation. Endothelial cells contribute by expressing the PGH2 import transporter slco2b1 and ptges3, the enzyme converting PGH2 into PGE2. Of note, differential niche cell expression of PGE2 biosynthesis enzymes is also observed in the mouse fetal liver. Taken altogether, our data suggest that the triad composed of neutrophils, macrophages, and endothelial cells sequentially and synergistically contributes to blood stem cell expansion during vertebrate development.
    Keywords:  HSCs; PGE2; hematopoietic niche; slco2b1
    DOI:  https://doi.org/10.15252/embj.2021108536
  20. Nat Commun. 2022 Aug 04. 13(1): 4520
    TP53-dependent toxicity of CRISPR/Cas9 cuts is differential across genomic loci and can confound genetic screening.
    Miguel M Álvarez, Josep Biayna, Fran Supek.
      CRISPR/Cas9 gene editing can inactivate genes in a precise manner. This process involves DNA double-strand breaks (DSB), which may incur a loss of cell fitness. We hypothesize that DSB toxicity may be variable depending on the chromatin environment in the targeted locus. Here, by analyzing isogenic cell line pair CRISPR experiments jointly with previous screening data from across ~900 cell lines, we show that TP53-associated break toxicity is higher in genomic regions that harbor active chromatin, such as gene regulatory elements or transcription elongation histone marks. DSB repair pathway choice and DNA sequence context also associate with toxicity. We also show that, due to noise introduced by differential toxicity of sgRNA-targeted sites, the power of genetic screens to detect conditional essentiality is reduced in TP53 wild-type cells. Understanding the determinants of Cas9 cut toxicity will help improve design of CRISPR reagents to avoid incidental selection of TP53-deficient and/or DNA repair deficient cells.
    DOI:  https://doi.org/10.1038/s41467-022-32285-1
  21. Nature. 2022 Aug 03.
    Transcriptome variation in human tissues revealed by long-read sequencing.
    Dafni A Glinos, Garrett Garborcauskas, Paul Hoffman, Nava Ehsan, Lihua Jiang, Alper Gokden, Xiaoguang Dai, François Aguet, Kathleen L Brown, Kiran Garimella, Tera Bowers, Maura Costello, Kristin Ardlie, Ruiqi Jian, Nathan R Tucker, Patrick T Ellinor, Eoghan D Harrington, Hua Tang, Michael Snyder, Sissel Juul, Pejman Mohammadi, Daniel G MacArthur, Tuuli Lappalainen, Beryl B Cummings.
      Regulation of transcript structure generates transcript diversity and plays an important role in human disease1-7. The advent of long-read sequencing technologies offers the opportunity to study the role of genetic variation in transcript structure8-16. In this Article, we present a large human long-read RNA-seq dataset using the Oxford Nanopore Technologies platform from 88 samples from Genotype-Tissue Expression (GTEx) tissues and cell lines, complementing the GTEx resource. We identified just over 70,000 novel transcripts for annotated genes, and validated the protein expression of 10% of novel transcripts. We developed a new computational package, LORALS, to analyse the genetic effects of rare and common variants on the transcriptome by allele-specific analysis of long reads. We characterized allele-specific expression and transcript structure events, providing new insights into the specific transcript alterations caused by common and rare genetic variants and highlighting the resolution gained from long-read data. We were able to perturb the transcript structure upon knockdown of PTBP1, an RNA binding protein that mediates splicing, thereby finding genetic regulatory effects that are modified by the cellular environment. Finally, we used this dataset to enhance variant interpretation and study rare variants leading to aberrant splicing patterns.
    DOI:  https://doi.org/10.1038/s41586-022-05035-y
  22. Leukemia. 2022 Jul 30.
    Droplet digital PCR for the detection of second-generation tyrosine kinase inhibitor-resistant BCR::ABL1 kinase domain mutations in chronic myeloid leukemia.
    Simona Soverini, Sara De Santis, Margherita Martelli, Cecilia Monaldi, Fausto Castagnetti, Gabriele Gugliotta, Cristina Papayannidis, Manuela Mancini, Samantha Bruno, Claudia Venturi, Katerina Machova Polakova, Thomas Ernst, Dianna Maar, Adam Corner, Michele Cavo.
      One of the indications for BCR::ABL1 mutation testing in chronic myeloid leukemia (CML) is when tyrosine kinase inhibitor therapy (TKI) needs to be changed for unsatisfactory response. In this study, we evaluated a droplet digital PCR (ddPCR)-based multiplex strategy for the detection and quantitation of transcripts harbouring mutations conferring resistance to second-generation TKIs (2GTKIs). Parallel quantitation of e13a2, e14a2 and e1a2 BCR::ABL1 fusion transcripts enables to express results as percentage of mutation positive- over total BCR::ABL1 transcripts. We determined the limit of blank in 60 mutation-negative samples. Accuracy was demonstrated by further analysis of 48 samples already studied by next generation sequencing (NGS). Mutations could be called down to 0.5% and across 3-logs of BCR::ABL1 levels. Retrospective review of BCR::ABL1 NGS results in 513 consecutive CML patients with non-optimal response to first- or second-line TKI therapy suggested that a ddPCR-based approach targeted against 2GTKI-resistant mutations would score samples as mutation-negative in 22% of patients with warning response to imatinib but only in 6% of patients with warning response to 2GTKIs. We conclude ddPCR represents an attractive method for easy, accurate and rapid screening for 2GTKI-resistant mutations impacting on TKI selection, although ddPCR cannot identify compound mutations.
    DOI:  https://doi.org/10.1038/s41375-022-01660-8
  23. Cell Stem Cell. 2022 Aug 04. pii: S1934-5909(22)00297-1. [Epub ahead of print]29(8): 1156-1158
    Turning the clock forward: Inflammation accelerates the aging of hematopoietic stem cells.
    Sarah S Burns, Reuben Kapur.
      In the current issue of Cell Stem Cell, Bogeska et al. demonstrate that repeated exposures to inflammation cause indelible and specific functional compromise and accelerated aging of long-term hematopoietic stem cells (LT-HSCs). This study proposes the notion that the cumulative inflammatory events over the course of an organism's lifespan may irreversibly damage LT-HSCs.
    DOI:  https://doi.org/10.1016/j.stem.2022.07.002
  24. Blood. 2022 Aug 02. pii: blood.2021015184. [Epub ahead of print]
    Dual-antigen targeted off-the-shelf NK cells show durable response and prevent antigen escape in lymphoma and leukemia.
    Frank Cichocki, Jode Goodridge, Ryan Bjordahl, Sajid Mahmood, Zachary B Davis, Svetlana Gaidarova, Ramzey Abujarour, Brian Groff, Alec Drake Witty, Hongbo Wang, Katie Tuininga, Behiye Kodal, Martin Felices, Gregory B Bonello, Janel Huffman, Thomas Dailey, Tom T Lee, Bruce Walcheck, Bahram Valamehr, Jeffrey S Miller.
      Substantial numbers of B cell leukemia and lymphoma patients relapse due to antigen loss or heterogeneity after anti-CD19 chimeric antigen receptor (CAR) T cell therapy. To overcome antigen escape and address antigen heterogeneity, we engineered induced pluripotent stem cell (iPSC)-derived NK cells to express both an NK cell-optimized anti-CD19 CAR for direct targeting and a high affinity, non-cleavable CD16 to augment antibody-dependent cellular cytotoxicity (ADCC). In addition, we introduced a membrane-bound IL-15/IL-15R fusion (IL-15RF) protein to promote in vivo persistence. These engineered cells, termed iDuo NK cells, displayed robust CAR-mediated cytotoxic activity that could be further enhanced with therapeutic antibodies targeting B cell malignancies. In multiple in vitro and xenogeneic adoptive transfer models, iDuo NK cells exhibited robust anti-lymphoma activity. Furthermore, iDuo NK cells effectively eliminated both CD19+ and CD19- lymphoma cells and displayed a unique propensity for targeting malignant cells over healthy cells that expressed CD19, features not achievable with anti-CAR19 T cells. iDuo NK cells combined with therapeutic antibodies represents a promising approach to prevent relapse due to antigen loss and tumor heterogeneity in patients with B cell malignancies.
    DOI:  https://doi.org/10.1182/blood.2021015184
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