bims-scepro 2024-06-09 papers

bims-scepro

Biomed News

on Stem cell proteostasis

Issue of 2024‒06‒09
23 papers selected by
William Grey, University of York

Advances in ex vivo expansion of hematopoietic stem and progenitor cells for clinical applications.
Sex-dependent niche responses modulate steady-state and regenerative hematopoiesis.
Clonal evolution of hematopoietic stem cells after cancer chemotherapy.
IκBα controls dormancy in hematopoietic stem cells via retinoic acid during embryonic development.
Near-perfect precise on-target editing of human hematopoietic stem and progenitor cells.
Targeted protein degradation systems to enhance Wnt signaling.
Single-cell transcriptomics reveal synergistic and antagonistic effects of T21 and GATA1s on hematopoiesis.
BRD4 degraders may effectively counteract therapeutic resistance of leukemic stem cells in AML and ALL.
Single-cell transcriptomic analysis of the immune microenvironment in pediatric acute leukemia.
MYCT1 controls environmental sensing in human haematopoietic stem cells.
Nucleotide depletion promotes cell fate transitions by inducing DNA replication stress.
Non-hierarchically related HSCs replenish platelets by distinct progenitor pathways.
Increasing Proteome Coverage Through a Reduction in Analyte Complexity in Single-Cell Equivalent Samples.
A CD36-dependent non-canonical lipid metabolism program promotes immune escape and resistance to hypomethylating agent therapy in AML.
OGA mutant aberrantly hydrolyzes O-GlcNAc modification from PDLIM7 to modulate p53 and cytoskeleton in promoting cancer cell malignancy.
Clonal hematopoiesis driven by mutated DNMT3A promotes inflammatory bone loss.
Reverse Phase Proteomic Array Profiling of Asparagine Synthetase Expression in Newly Diagnosed Acute Myeloid Leukemia.
Scanorama: integrating large and diverse single-cell transcriptomic datasets.
ROS-mediated cytoplasmic localization of CARM1 induces mitochondrial fission through DRP1 methylation.
Orthogonal proteogenomic analysis identifies the druggable PA2G4-MYC axis in 3q26 AML.
Determining the ERK-regulated phosphoproteome driving KRAS-mutant cancer.
PROteolysis-Targeting Chimeras (PROTACs) in leukemia: overview and future perspectives.
Epigenetic and proteomic signatures associate with clonal hematopoiesis expansion rate.

Front Bioeng Biotechnol. 2024 ;12 1380950

Advances in ex vivo expansion of hematopoietic stem and progenitor cells for clinical applications.

André Branco, Janakiram Rayabaram, Cláudia C Miranda, Ana Fernandes-Platzgummer, Tiago G Fernandes, Suchitra Sajja, Cláudia L da Silva, Mohan C Vemuri.

  As caretakers of the hematopoietic system, hematopoietic stem cells assure a lifelong supply of differentiated populations that are responsible for critical bodily functions, including oxygen transport, immunological protection and coagulation. Due to the far-reaching influence of the hematopoietic system, hematological disorders typically have a significant impact on the lives of individuals, even becoming fatal. Hematopoietic cell transplantation was the first effective therapeutic avenue to treat such hematological diseases. Since then, key use and manipulation of hematopoietic stem cells for treatments has been aspired to fully take advantage of such an important cell population. Limited knowledge on hematopoietic stem cell behavior has motivated in-depth research into their biology. Efforts were able to uncover their native environment and characteristics during development and adult stages. Several signaling pathways at a cellular level have been mapped, providing insight into their machinery. Important dynamics of hematopoietic stem cell maintenance were begun to be understood with improved comprehension of their metabolism and progressive aging. These advances have provided a solid platform for the development of innovative strategies for the manipulation of hematopoietic stem cells. Specifically, expansion of the hematopoietic stem cell pool has triggered immense interest, gaining momentum. A wide range of approaches have sprouted, leading to a variety of expansion systems, from simpler small molecule-based strategies to complex biomimetic scaffolds. The recent approval of Omisirge, the first expanded hematopoietic stem and progenitor cell product, whose expansion platform is one of the earliest, is predictive of further successes that might arise soon. In order to guarantee the quality of these ex vivo manipulated cells, robust assays that measure cell function or potency need to be developed. Whether targeting hematopoietic engraftment, immunological differentiation potential or malignancy clearance, hematopoietic stem cells and their derivatives need efficient scaling of their therapeutic potency. In this review, we comprehensively view hematopoietic stem cells as therapeutic assets, going from fundamental to translational.

Keywords:  clinical translation; ex vivo expansion; hematopoietic stem and progenitor cells; metabolism; potency; signaling

DOI:  https://doi.org/10.3389/fbioe.2024.1380950
Exp Hematol. 2024 Jun 05. pii: S0301-472X(24)00106-1. [Epub ahead of print] 104247

Sex-dependent niche responses modulate steady-state and regenerative hematopoiesis.

Rahul Chaudhary, Julianne N P Smith, Riya Tiwari, Bailey R Klein, Brittany A Cordova, Frederick Petroze, Brian Richardson, Alyssia V Broncano, Juyeun Lee, Prerana Bangalore Parthasarathy, Karina Inacio Ladislau De Carvalho, Scott J Cameron, Justin D Lathia, Wendy A Goodman, Mark J Cameron, Amar B Desai.

  Hematopoietic stem cells (HSCs) adapt to organismal blood production needs by balancing self-renewal and differentiation, adjusting to physiological demands and external stimuli. While sex differences have been implicated in differential hematopoietic function in males vs. females, the mediators responsible for these effects require further study. Here, we characterize hematopoiesis at steady state and during regeneration following hematopoietic stem cell transplantation (HST). RNA sequencing of lineage(-) bone marrow cells from C57/Bl6 mice revealed a broad transcriptional similarity between the sexes. However, we identified distinct sex differences in key biological pathways, with female cells showing reduced expression of signatures involved in inflammation and an enrichment of genes related to glycolysis, hypoxia, and cell cycle regulation, suggesting a more quiescent and less inflammatory profile compared to male cells. To determine the functional impacts of the observed transcriptomic differences, we performed sex-matched and mismatched transplantation studies of lineage(-) donor cells. During short-term 56-day HST recovery we found a male donor cell proliferative advantage, coinciding with elevated serum TNF-α, and a male recipient engraftment advantage, coinciding with increased serum CXCL12. Together, we show that sex-specific cell responses, marked by differing expression of pathways regulating metabolism, hypoxia, and inflammation, shapes normal and regenerative hematopoiesis, with implications for the clinical understanding of hematopoietic function.

Keywords:  hematopoiesis; hematopoietic stem cell transplantation; inflammation; sex differences; stem cells

DOI:  https://doi.org/10.1016/j.exphem.2024.104247
bioRxiv. 2024 May 24. pii: 2024.05.23.595594. [Epub ahead of print]

Clonal evolution of hematopoietic stem cells after cancer chemotherapy.

Hidetaka Uryu, Koichi Saeki, Hiroshi Haeno, Chiraag Deepak Kapadia, Ken Furudate, Jyoti Nangalia, Michael Spencer Chapman, Li Zhao, Joanne I Hsu, Chong Zhao, Shujuan Chen, Tomoyuki Tanaka, Zongrui Li, Hui Yang, Courtney DiNardo, Naval Daver, Naveen Pemmaraju, Nitin Jain, Farhad Ravandi, Jianhua Zhang, Xingzhi Song, Erika Thompson, Hongli Tang, Latasha Little, Curtis Gumbs, Robert Z Orlowski, Muzaffar Qazilbash, Kapil Bhalla, Simona Colla, Hagop Kantarjian, Rashmi Kanagal Shamanna, Carlos Bueso- Ramos, Daisuke Nakada, P Andrew Futreal, Elizabeth Shpall, Margaret Goodell, Guillermo Garcia-Manero, Koichi Takahashi.

Normal hematopoietic stem and progenitor cells (HSPCs) inherently accumulate somatic mutations and lose clonal diversity with age, processes implicated in the development of myeloid malignancies 1 . The impact of exogenous stressors, such as cancer chemotherapies, on the genomic integrity and clonal dynamics of normal HSPCs is not well defined. We conducted whole-genome sequencing on 1,032 single-cell-derived HSPC colonies from 10 patients with multiple myeloma (MM), who had undergone various chemotherapy regimens. Our findings reveal that melphalan treatment distinctly increases mutational burden with a unique mutation signature, whereas other MM chemotherapies do not significantly affect the normal mutation rate of HSPCs. Among these therapy-induced mutations were several oncogenic drivers such as TET2 and PPM1D . Phylogenetic analysis showed a clonal architecture in post-treatment HSPCs characterized by extensive convergent evolution of mutations in genes such as TP53 and PPM1D . Consequently, the clonal diversity and structure of post-treatment HSPCs mirror those observed in normal elderly individuals, suggesting an accelerated clonal aging due to chemotherapy. Furthermore, analysis of matched therapy-related myeloid neoplasm (t-MN) samples, which occurred 1-8 years later, enabled us to trace the clonal origin of t-MNs to a single HSPC clone among a group of clones with competing malignant potential, indicating the critical role of secondary mutations in dictating clonal dominance and malignant transformation. Our findings suggest that cancer chemotherapy promotes an oligoclonal architecture with multiple HSPC clones possessing competing leukemic potentials, setting the stage for the selective emergence of a singular clone that evolves into t-MNs after acquiring secondary mutations. These results underscore the importance of further systematic research to elucidate the long-term hematological consequences of cancer chemotherapy.

DOI: https://doi.org/10.1101/2024.05.23.595594
Nat Commun. 2024 Jun 01. 15(1): 4673

IκBα controls dormancy in hematopoietic stem cells via retinoic acid during embryonic development.

Roshana Thambyrajah, Maria Maqueda, Muhammad Zaki Fadlullah, Martin Proffitt, Wen Hao Neo, Yolanda Guillén, Marta Casado-Pelaez, Patricia Herrero-Molinero, Carla Brujas, Noemi Castelluccio, Jessica González, Arnau Iglesias, Laura Marruecos, Cristina Ruiz-Herguido, Manel Esteller, Elisabetta Mereu, Georges Lacaud, Lluis Espinosa, Anna Bigas.

Recent findings suggest that Hematopoietic Stem Cells (HSC) and progenitors arise simultaneously and independently of each other already in the embryonic aorta-gonad mesonephros region, but it is still unknown how their different features are established. Here, we uncover IκBα (Nfkbia, the inhibitor of NF-κB) as a critical regulator of HSC proliferation throughout development. IκBα balances retinoic acid signaling levels together with the epigenetic silencer, PRC2, specifically in HSCs. Loss of IκBα decreases proliferation of HSC and induces a dormancy related gene expression signature instead. Also, IκBα deficient HSCs respond with superior activation to in vitro culture and in serial transplantation. At the molecular level, chromatin regions harboring binding motifs for retinoic acid signaling are hypo-methylated for the PRC2 dependent H3K27me3 mark in IκBα deficient HSCs. Overall, we show that the proliferation index in the developing HSCs is regulated by a IκBα-PRC2 axis, which controls retinoic acid signaling.

DOI: https://doi.org/10.1038/s41467-024-48854-5
Elife. 2024 Jun 03. pii: RP91288. [Epub ahead of print]12

Near-perfect precise on-target editing of human hematopoietic stem and progenitor cells.

Fanny-Mei Cloarec-Ung, Jamie Beaulieu, Arunan Suthananthan, Bernhard Lehnertz, Guy Sauvageau, Hilary M Sheppard, David J H F Knapp.

  Precision gene editing in primary hematopoietic stem and progenitor cells (HSPCs) would facilitate both curative treatments for monogenic disorders as well as disease modelling. Precise efficiencies even with the CRISPR/Cas system, however, remain limited. Through an optimization of guide RNA delivery, donor design, and additives, we have now obtained mean precise editing efficiencies >90% on primary cord blood HSCPs with minimal toxicity and without observed off-target editing. The main protocol modifications needed to achieve such high efficiencies were the addition of the DNA-PK inhibitor AZD7648, and the inclusion of spacer-breaking silent mutations in the donor in addition to mutations disrupting the PAM sequence. Critically, editing was even across the progenitor hierarchy, did not substantially distort the hierarchy or affect lineage outputs in colony-forming cell assays or the frequency of high self-renewal potential long-term culture initiating cells. As modelling of many diseases requires heterozygosity, we also demonstrated that the overall editing and zygosity can be tuned by adding in defined mixtures of mutant and wild-type donors. With these optimizations, editing at near-perfect efficiency can now be accomplished directly in human HSPCs. This will open new avenues in both therapeutic strategies and disease modelling.

Keywords:  CRISPR; genome editing; hematopoiesis; hematopoietic stem cell; human; leukemia; regenerative medicine; stem cell; stem cells

DOI:  https://doi.org/10.7554/eLife.91288
Elife. 2024 Jun 07. pii: RP93908. [Epub ahead of print]13

Targeted protein degradation systems to enhance Wnt signaling.

Parthasarathy Sampathkumar, Heekyung Jung, Hui Chen, Zhengjian Zhang, Nicholas Suen, Yiran Yang, Zhong Huang, Tom Lopez, Robert Benisch, Sung-Jin Lee, Jay Ye, Wen-Chen Yeh, Yang Li.

  Molecules that facilitate targeted protein degradation (TPD) offer great promise as novel therapeutics. The human hepatic lectin asialoglycoprotein receptor (ASGR) is selectively expressed on hepatocytes. We have previously engineered an anti-ASGR1 antibody-mutant RSPO2 (RSPO2RA) fusion protein (called SWEETS) to drive tissue-specific degradation of ZNRF3/RNF43 E3 ubiquitin ligases, which achieved hepatocyte-specific enhanced Wnt signaling, proliferation, and restored liver function in mouse models, and an antibody-RSPO2RA fusion molecule is currently in human clinical trials. In the current study, we identified two new ASGR1- and ASGR1/2-specific antibodies, 8M24 and 8G8. High-resolution crystal structures of ASGR1:8M24 and ASGR2:8G8 complexes revealed that these antibodies bind to distinct epitopes on opposing sides of ASGR, away from the substrate-binding site. Both antibodies enhanced Wnt activity when assembled as SWEETS molecules with RSPO2RA through specific effects sequestering E3 ligases. In addition, 8M24-RSPO2RA and 8G8-RSPO2RA efficiently downregulate ASGR1 through TPD mechanisms. These results demonstrate the possibility of combining different therapeutic effects and degradation mechanisms in a single molecule.

Keywords:  ASGR1; LYTAC; PROTAC; R-spondin; SWEETS; Wnt; biochemistry; chemical biology; none; regenerative medicine; stem cells

DOI:  https://doi.org/10.7554/eLife.93908
bioRxiv. 2024 May 25. pii: 2024.05.24.595827. [Epub ahead of print]

Single-cell transcriptomics reveal synergistic and antagonistic effects of T21 and GATA1s on hematopoiesis.

Kaoru Takasaki, Eric K Wafula, Sara S Kumar, David Smith, Alyssa L Gagne, Deborah L French, Christopher S Thom, Stella T Chou.

Trisomy 21 (T21), or Down syndrome (DS), is associated with baseline macrocytic erythrocytosis, thrombocytopenia, and neutrophilia, and transient abnormal myelopoiesis (TAM) and myeloid leukemia of DS (ML-DS). TAM and ML-DS blasts both arise from an aberrant megakaryocyte-erythroid progenitor and exclusively express GATA1s, the truncated isoform of GATA1 , while germline GATA1s mutations in a non-T21 context lead to congenital cytopenias without a leukemic predisposition. This suggests that T21 and GATA1s perturb hematopoiesis independently and synergistically, but this interaction has been challenging to study in part due to limited human cell and murine models. To dissect the developmental impacts of GATA1s on hematopoiesis in euploid and T21 cells, we performed a single-cell RNA-sequencing timecourse on hematopoietic progenitors (HPCs) derived from isogenic human induced pluripotent stem cells differing only by chromosome 21 and/or GATA1 status. These HPCs were surprisingly heterogeneous and displayed spontaneous lineage skew apparently dictated by T21 and/or GATA1s. In euploid cells, GATA1s nearly eliminated erythropoiesis, impaired MK maturation, and promoted an immature myelopoiesis, while in T21 cells, GATA1s appeared to compete with the enhanced erythropoiesis and suppressed megakaryopoiesis driven by T21 to give rise to immature erythrocytes, MKs, and myeloid cells. T21 and GATA1s both disrupted temporal regulation of lineage-specific transcriptional programs and specifically perturbed cell cycle genes. These findings in an isogenic system can thus be attributed specifically to T21 and GATA1s and suggest that these genetic changes together enhance HPC proliferation at the expense of maturation, consistent with a pro-leukemic phenotype.

DOI: https://doi.org/10.1101/2024.05.24.595827
Am J Hematol. 2024 Jun 01.

BRD4 degraders may effectively counteract therapeutic resistance of leukemic stem cells in AML and ALL.

Karin Bauer, Alexander Hauswirth, Karoline V Gleixner, Georg Greiner, Johannes Thaler, Peter Bettelheim, Yüksel Filik, Elisabeth Koller, Gregor Hoermann, Philipp B Staber, Wolfgang R Sperr, Felix Keil, Peter Valent.

Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) are life-threatening hematopoietic malignancies characterized by clonal expansion of leukemic blasts in the bone marrow and peripheral blood. The epigenetic reader BRD4 and its downstream effector MYC have recently been identified as potential drug targets in human AML and ALL. We compared anti-leukemic efficacies of the small-molecule BET inhibitor JQ1 and the recently developed BRD4 degraders dBET1 and dBET6 in AML and ALL cells. JQ1, dBET1, and dBET6 were found to suppress growth and viability in all AML and ALL cell lines examined as well as in primary patient-derived AML and ALL cells, including CD34+/CD38- and CD34+/CD38+ leukemic stem and progenitor cells, independent of the type (variant) of leukemia or molecular driver expressed in leukemic cells. Moreover, we found that dBET6 overcomes osteoblast-induced drug resistance in AML and ALL cells, regardless of the type of leukemia or the drug applied. Most promising cooperative or even synergistic drug combination effects were seen with dBET6 and the FLT3 ITD blocker gilteritinib in FLT3 ITD-mutated AML cells, and with dBET6 and the multi-kinase blocker ponatinib in BCR::ABL1+ ALL cells. Finally, all BRD4-targeting drugs suppressed interferon-gamma- and tumor necrosis factor-alpha-induced expression of the resistance-related checkpoint antigen PD-L1 in AML and ALL cells, including LSC. In all assays examined, the BRD4 degrader dBET6 was a superior anti-leukemic drug compared with dBET1 and JQ1. Together, BRD4 degraders may provide enhanced inhibition of multiple mechanisms of therapy resistance in AML and ALL.

DOI: https://doi.org/10.1002/ajh.27385
Cancer Lett. 2024 Jun 04. pii: S0304-3835(24)00412-9. [Epub ahead of print] 217018

Single-cell transcriptomic analysis of the immune microenvironment in pediatric acute leukemia.

Jiapei Yuan, Jingliao Zhang, Beibei Zhao, Fang Liu, Tianfeng Liu, Yongjuan Duan, Yumei Chen, Xiaojuan Chen, Yao Zou, Li Zhang, Ye Guo, Wenyu Yang, Yang Yang, Jun Wei, Xiaofan Zhu, Yingchi Zhang.

  Relapse and treatment resistance pose significant challenges in the management of pediatric B cell acute lymphoblastic leukemia (B-ALL) and acute myeloid leukemia (AML). The efficacy of immunotherapy in leukemia remains limited due to factors such as the immunosuppressive tumor microenvironment (TME) and lack of suitable immunotherapeutic targets. Thus, an in-depth characterization of the TME in pediatric leukemia is warranted to improve the efficacy of immunotherapy. Here, we used single-cell RNA sequencing (scRNA-seq) to characterize the TME of pediatric B-ALL and AML, focusing specifically on bone-marrow-derived T cells. Moreover, we investigated the transcriptome changes during the initiation, remission, and relapse stages of pediatric AML. Our findings revealed that specific functional expression programs correlated with fluctuations in various T cell subsets, which may be associated with AML progression and relapse. Furthermore, our analysis of cellular communication networks led to the identification of VISTA, CD244, and TIM3 as potential immunotherapeutic targets in pediatric AML. Finally, we detected elevated proportions of γδ T cells and associated functional genes in samples from pediatric patients diagnosed with B-ALL and AML, which could inform the development of novel therapeutic approaches, potentially focusing on γδ T cells.

Keywords:  Pediatric acute leukemia; T cells; Tumor microenvironment; immunotherapeutic targets; γδ T cells

DOI:  https://doi.org/10.1016/j.canlet.2024.217018
Nature. 2024 Jun 05.

MYCT1 controls environmental sensing in human haematopoietic stem cells.

Júlia Aguadé-Gorgorió, Yasaman Jami-Alahmadi, Vincenzo Calvanese, Maya Kardouh, Iman Fares, Haley Johnson, Valerie Rezek, Feiyang Ma, Mattias Magnusson, Yanling Wang, Juliana E Shin, Karina J Nance, Helen S Goodridge, Simone Liebscher, Katja Schenke-Layland, Gay M Crooks, James A Wohlschlegel, Hanna K A Mikkola.

The processes that govern human haematopoietic stem cell (HSC) self-renewal and engraftment are poorly understood and challenging to recapitulate in culture to reliably expand functional HSCs1-3. Here we identify MYC target 1 (MYCT1; also known as MTLC) as a crucial human HSC regulator that moderates endocytosis and environmental sensing in HSCs. MYCT1 is selectively expressed in undifferentiated human haematopoietic stem and progenitor cells (HSPCs) and endothelial cells but becomes markedly downregulated during HSC culture. Lentivirus-mediated knockdown of MYCT1 prevented human fetal liver and cord blood (CB) HSPC expansion and engraftment. By contrast, restoring MYCT1 expression improved the expansion and engraftment of cultured CB HSPCs. Single-cell RNA sequencing of human CB HSPCs in which MYCT1 was knocked down or overexpressed revealed that MYCT1 governs important regulatory programmes and cellular properties essential for HSC stemness, such as ETS factor expression and low mitochondrial activity. MYCT1 is localized in the endosomal membrane in HSPCs and interacts with vesicle trafficking regulators and signalling machinery. MYCT1 loss in HSPCs led to excessive endocytosis and hyperactive signalling responses, whereas restoring MYCT1 expression balanced culture-induced endocytosis and dysregulated signalling. Moreover, sorting cultured CB HSPCs on the basis of lowest endocytosis rate identified HSPCs with preserved MYCT1 expression and MYCT1-regulated HSC stemness programmes. Our work identifies MYCT1-moderated endocytosis and environmental sensing as essential regulatory mechanisms required to preserve human HSC stemness. Our data also pinpoint silencing of MYCT1 as a cell-culture-induced vulnerability that compromises human HSC expansion.

DOI: https://doi.org/10.1038/s41586-024-07478-x
Dev Cell. 2024 May 30. pii: S1534-5807(24)00327-7. [Epub ahead of print]

Nucleotide depletion promotes cell fate transitions by inducing DNA replication stress.

Brian T Do, Peggy P Hsu, Sidney Y Vermeulen, Zhishan Wang, Taghreed Hirz, Keene L Abbott, Najihah Aziz, Joseph M Replogle, Stefan Bjelosevic, Jonathan Paolino, Samantha A Nelson, Samuel Block, Alicia M Darnell, Raphael Ferreira, Hanyu Zhang, Jelena Milosevic, Daniel R Schmidt, Christopher Chidley, Isaac S Harris, Jonathan S Weissman, Yana Pikman, Kimberly Stegmaier, Sihem Cheloufi, Xiaofeng A Su, David B Sykes, Matthew G Vander Heiden.

  Control of cellular identity requires coordination of developmental programs with environmental factors such as nutrient availability, suggesting that perturbing metabolism can alter cell state. Here, we find that nucleotide depletion and DNA replication stress drive differentiation in human and murine normal and transformed hematopoietic systems, including patient-derived acute myeloid leukemia (AML) xenografts. These cell state transitions begin during S phase and are independent of ATR/ATM checkpoint signaling, double-stranded DNA break formation, and changes in cell cycle length. In systems where differentiation is blocked by oncogenic transcription factor expression, replication stress activates primed regulatory loci and induces lineage-appropriate maturation genes despite the persistence of progenitor programs. Altering the baseline cell state by manipulating transcription factor expression causes replication stress to induce genes specific for alternative lineages. The ability of replication stress to selectively activate primed maturation programs across different contexts suggests a general mechanism by which changes in metabolism can promote lineage-appropriate cell state transitions.

Keywords:  cancer; cell fate; cell state; dependencies; differentiation; epigenetics; hematopoiesis; metabolism; nucleotides; replication; replication stress

DOI:  https://doi.org/10.1016/j.devcel.2024.05.010
Nat Immunol. 2024 Jun;25(6): 955-956

Non-hierarchically related HSCs replenish platelets by distinct progenitor pathways.

DOI: https://doi.org/10.1038/s41590-024-01878-x
J Proteome Res. 2024 Jun 04.

Increasing Proteome Coverage Through a Reduction in Analyte Complexity in Single-Cell Equivalent Samples.

Marion Pang, Jeff J Jones, Ting-Yu Wang, Baiyi Quan, Nicole J Kubat, Yanping Qiu, Michael L Roukes, Tsui-Fen Chou.

  The advancement of sophisticated instrumentation in mass spectrometry has catalyzed an in-depth exploration of complex proteomes. This exploration necessitates a nuanced balance in experimental design, particularly between quantitative precision and the enumeration of analytes detected. In bottom-up proteomics, a key challenge is that oversampling of abundant proteins can adversely affect the identification of a diverse array of unique proteins. This issue is especially pronounced in samples with limited analytes, such as small tissue biopsies or single-cell samples. Methods such as depletion and fractionation are suboptimal to reduce oversampling in single cell samples, and other improvements on LC and mass spectrometry technologies and methods have been developed to address the trade-off between precision and enumeration. We demonstrate that by using a monosubstrate protease for proteomic analysis of single-cell equivalent digest samples, an improvement in quantitative accuracy can be achieved, while maintaining high proteome coverage established by trypsin. This improvement is particularly vital for the field of single-cell proteomics, where single-cell samples with limited number of protein copies, especially in the context of low-abundance proteins, can benefit from considering analyte complexity. Considerations about analyte complexity, alongside chromatographic complexity, integration with data acquisition methods, and other factors such as those involving enzyme kinetics, will be crucial in the design of future single-cell workflows.

Keywords:  bottom-up proteomics; peptide identification optimization; protease choice; single-cell proteomics

DOI:  https://doi.org/10.1021/acs.jproteome.4c00062
Cell Rep Med. 2024 May 29. pii: S2666-3791(24)00284-2. [Epub ahead of print] 101592

A CD36-dependent non-canonical lipid metabolism program promotes immune escape and resistance to hypomethylating agent therapy in AML.

He-Zhou Guo, Rui-Xue Feng, Yan-Jie Zhang, Ye-Hua Yu, Wei Lu, Jia-Jia Liu, Shao-Xin Yang, Chong Zhao, Zhao-Li Zhang, Shan-He Yu, Hui Jin, Si-Xuan Qian, Jian-Yong Li, Jiang Zhu, Jun Shi.

Environmental lipids are essential for fueling tumor energetics, but whether these exogenous lipids transported into cancer cells facilitate immune escape remains unclear. Here, we find that CD36, a transporter for exogenous lipids, promotes acute myeloid leukemia (AML) immune evasion. We show that, separately from its established role in lipid oxidation, CD36 on AML cells senses oxidized low-density lipoprotein (OxLDL) to prime the TLR4-LYN-MYD88-nuclear factor κB (NF-κB) pathway, and exogenous palmitate transfer via CD36 further potentiates this innate immune pathway by supporting ZDHHC6-mediated MYD88 palmitoylation. Subsequently, NF-κB drives the expression of immunosuppressive genes that inhibit anti-tumor T cell responses. Notably, high-fat-diet or hypomethylating agent decitabine treatment boosts the immunosuppressive potential of AML cells by hijacking CD36-dependent innate immune signaling, leading to a dampened therapeutic effect. This work is of translational interest because lipid restriction by US Food and Drug Administration (FDA)-approved lipid-lowering statin drugs improves the efficacy of decitabine therapy by weakening leukemic CD36-mediated immunosuppression.

DOI: https://doi.org/10.1016/j.xcrm.2024.101592
Proc Natl Acad Sci U S A. 2024 Jun 11. 121(24): e2320867121

OGA mutant aberrantly hydrolyzes O-GlcNAc modification from PDLIM7 to modulate p53 and cytoskeleton in promoting cancer cell malignancy.

Chia-Wei Hu, Ao Wang, Dacheng Fan, Matthew Worth, Zhengwei Chen, Junfeng Huang, Jinshan Xie, John Macdonald, Lingjun Li, Jiaoyang Jiang.

  O-GlcNAcase (OGA) is the only human enzyme that catalyzes the hydrolysis (deglycosylation) of O-linked beta-N-acetylglucosaminylation (O-GlcNAcylation) from numerous protein substrates. OGA has broad implications in many challenging diseases including cancer. However, its role in cell malignancy remains mostly unclear. Here, we report that a cancer-derived point mutation on the OGA's noncatalytic stalk domain aberrantly modulates OGA interactome and substrate deglycosylation toward a specific set of proteins. Interestingly, our quantitative proteomic studies uncovered that the OGA stalk domain mutant preferentially deglycosylated protein substrates with +2 proline in the sequence relative to the O-GlcNAcylation site. One of the most dysregulated substrates is PDZ and LIM domain protein 7 (PDLIM7), which is associated with the tumor suppressor p53. We found that the aberrantly deglycosylated PDLIM7 suppressed p53 gene expression and accelerated p53 protein degradation by promoting the complex formation with E3 ubiquitin ligase MDM2. Moreover, deglycosylated PDLIM7 significantly up-regulated the actin-rich membrane protrusions on the cell surface, augmenting the cancer cell motility and aggressiveness. These findings revealed an important but previously unappreciated role of OGA's stalk domain in protein substrate recognition and functional modulation during malignant cell progression.

Keywords:  O-GlcNAcase; O-GlcNAcylation; PDLIM7; cancer; deglycosylation

DOI:  https://doi.org/10.1073/pnas.2320867121
Cell. 2024 May 30. pii: S0092-8674(24)00492-6. [Epub ahead of print]

Clonal hematopoiesis driven by mutated DNMT3A promotes inflammatory bone loss.

Hui Wang, Kimon Divaris, Bohu Pan, Xiaofei Li, Jong-Hyung Lim, Gundappa Saha, Marko Barovic, Danai Giannakou, Jonathan M Korostoff, Yu Bing, Souvik Sen, Kevin Moss, Di Wu, James D Beck, Christie M Ballantyne, Pradeep Natarajan, Kari E North, Mihai G Netea, Triantafyllos Chavakis, George Hajishengallis.

  Clonal hematopoiesis of indeterminate potential (CHIP) arises from aging-associated acquired mutations in hematopoietic progenitors, which display clonal expansion and produce phenotypically altered leukocytes. We associated CHIP-DNMT3A mutations with a higher prevalence of periodontitis and gingival inflammation among 4,946 community-dwelling adults. To model DNMT3A-driven CHIP, we used mice with the heterozygous loss-of-function mutation R878H, equivalent to the human hotspot mutation R882H. Partial transplantation with Dnmt3aR878H/+ bone marrow (BM) cells resulted in clonal expansion of mutant cells into both myeloid and lymphoid lineages and an elevated abundance of osteoclast precursors in the BM and osteoclastogenic macrophages in the periphery. DNMT3A-driven clonal hematopoiesis in recipient mice promoted naturally occurring periodontitis and aggravated experimentally induced periodontitis and arthritis, associated with enhanced osteoclastogenesis, IL-17-dependent inflammation and neutrophil responses, and impaired regulatory T cell immunosuppressive activity. DNMT3A-driven clonal hematopoiesis and, subsequently, periodontitis were suppressed by rapamycin treatment. DNMT3A-driven CHIP represents a treatable state of maladaptive hematopoiesis promoting inflammatory bone loss.

Keywords:  DNMT3A; T cells; arthritis; bone marrow; clonal hematopoiesis of indeterminate potential; hematopoietic stem and progenitor cells; inflammation; neutrophils; osteoclastogenesis; periodontitis

DOI:  https://doi.org/10.1016/j.cell.2024.05.003
J Proteome Res. 2024 Jun 03.

Reverse Phase Proteomic Array Profiling of Asparagine Synthetase Expression in Newly Diagnosed Acute Myeloid Leukemia.

Nisha Narayanan, Jennifer Marvin-Peek, Mohamad K Abouelnaaj, Dhabya Majid, Bofei Wang, Brandon D Brown, Yihua Qiu, Steven M Kornblau, Hussein A Abbas.

  Asparaginase-based therapy is a cornerstone in acute lymphoblastic leukemia (ALL) treatment, capitalizing on the methylation status of the asparagine synthetase (ASNS) gene, which renders ALL cells reliant on extracellular asparagine. Contrastingly, ASNS expression in acute myeloid leukemia (AML) has not been thoroughly investigated, despite studies suggesting that AML with chromosome 7/7q deletions might have reduced ASNS levels. Here, we leverage reverse phase protein arrays to measure ASNS expression in 810 AML patients and assess its impact on outcomes. We find that AML with inv(16) has the lowest overall ASNS expression. While AML with deletion 7/7q had ASNS levels slightly lower than those of AML without deletion 7/7q, this observation was not significant. Low ASNS expression correlated with improved overall survival (46 versus 54 weeks, respectively, p = 0.011), whereas higher ASNS levels were associated with better response to venetoclax-based therapy. Protein correlation analysis demonstrated association between ASNS and proteins involved in methylation and DNA repair. In conclusion, while ASNS expression was not lower in patients with deletion 7/7q as initially predicted, ASNS levels were highly variable across AML patients. Further studies are needed to assess whether patients with low ASNS expression are susceptible to asparaginase-based therapy due to their inability to augment compensatory ASNS expression upon asparagine depletion.

Keywords:  acute myeloid leukemia (AML); asparagine synthetase (ASNS); chromosome 7/7q deletion; reverse phase proteomics array (RPPA); venetoclax

DOI:  https://doi.org/10.1021/acs.jproteome.4c00130
Nat Protoc. 2024 Jun 06.

Scanorama: integrating large and diverse single-cell transcriptomic datasets.

Brian L Hie, Soochi Kim, Thomas A Rando, Bryan Bryson, Bonnie Berger.

Merging diverse single-cell RNA sequencing (scRNA-seq) data from numerous experiments, laboratories and technologies can uncover important biological insights. Nonetheless, integrating scRNA-seq data encounters special challenges when the datasets are composed of diverse cell type compositions. Scanorama offers a robust solution for improving the quality and interpretation of heterogeneous scRNA-seq data by effectively merging information from diverse sources. Scanorama is designed to address the technical variation introduced by differences in sample preparation, sequencing depth and experimental batches that can confound the analysis of multiple scRNA-seq datasets. Here we provide a detailed protocol for using Scanorama within a Scanpy-based single-cell analysis workflow coupled with Google Colaboratory, a cloud-based free Jupyter notebook environment service. The protocol involves Scanorama integration, a process that typically spans 0.5-3 h. Scanorama integration requires a basic understanding of cellular biology, transcriptomic technologies and bioinformatics. Our protocol and new Scanorama-Colaboratory resource should make scRNA-seq integration more widely accessible to researchers.

DOI: https://doi.org/10.1038/s41596-024-00991-3
Redox Biol. 2024 May 31. pii: S2213-2317(24)00190-3. [Epub ahead of print]73 103212

ROS-mediated cytoplasmic localization of CARM1 induces mitochondrial fission through DRP1 methylation.

Yena Cho, Yong Kee Kim.

  The dynamic regulation of mitochondria through fission and fusion is essential for maintaining cellular homeostasis. In this study, we discovered a role of coactivator-associated arginine methyltransferase 1 (CARM1) in mitochondrial dynamics. CARM1 methylates specific residues (R403 and R634) on dynamin-related protein 1 (DRP1). Methylated DRP1 interacts with mitochondrial fission factor (Mff) and forms self-assembly on the outer mitochondrial membrane, thereby triggering fission, reducing oxygen consumption, and increasing reactive oxygen species (ROS) production. This sets in motion a feedback loop that facilitates the translocation of CARM1 from the nucleus to the cytoplasm, enhancing DRP1 methylation and ROS production through mitochondrial fragmentation. Consequently, ROS reinforces the CARM1-DRP1-ROS axis, resulting in cellular senescence. Depletion of CARM1 or DRP1 impedes cellular senescence by reducing ROS accumulation. The uncovering of the above-described mechanism fills a missing piece in the vicious cycle of ROS-induced senescence and contributes to a better understanding of the aging process.

Keywords:  CARM1; DRP1; Methylation; Mitochondrial dynamics; ROS; Senescence

DOI:  https://doi.org/10.1016/j.redox.2024.103212
Nat Commun. 2024 Jun 04. 15(1): 4739

Orthogonal proteogenomic analysis identifies the druggable PA2G4-MYC axis in 3q26 AML.

Matteo Marchesini, Andrea Gherli, Elisa Simoncini, Lucas Moron Dalla Tor, Anna Montanaro, Natthakan Thongon, Federica Vento, Chiara Liverani, Elisa Cerretani, Anna D'Antuono, Luca Pagliaro, Raffaella Zamponi, Chiara Spadazzi, Elena Follini, Benedetta Cambò, Mariateresa Giaimo, Angela Falco, Gabriella Sammarelli, Giannalisa Todaro, Sabrina Bonomini, Valentina Adami, Silvano Piazza, Claudia Corbo, Bruno Lorusso, Federica Mezzasoma, Costanza Anna Maria Lagrasta, Maria Paola Martelli, Roberta La Starza, Antonio Cuneo, Franco Aversa, Cristina Mecucci, Federico Quaini, Simona Colla, Giovanni Roti.

The overexpression of the ecotropic viral integration site-1 gene (EVI1/MECOM) marks the most lethal acute myeloid leukemia (AML) subgroup carrying chromosome 3q26 abnormalities. By taking advantage of the intersectionality of high-throughput cell-based and gene expression screens selective and pan-histone deacetylase inhibitors (HDACis) emerge as potent repressors of EVI1. To understand the mechanism driving on-target anti-leukemia activity of this compound class, here we dissect the expression dynamics of the bone marrow leukemia cells of patients treated with HDACi and reconstitute the EVI1 chromatin-associated co-transcriptional complex merging on the role of proliferation-associated 2G4 (PA2G4) protein. PA2G4 overexpression rescues AML cells from the inhibitory effects of HDACis, while genetic and small molecule inhibition of PA2G4 abrogates EVI1 in 3q26 AML cells, including in patient-derived leukemia xenografts. This study positions PA2G4 at the crosstalk of the EVI1 leukemogenic signal for developing new therapeutics and urges the use of HDACis-based combination therapies in patients with 3q26 AML.

DOI: https://doi.org/10.1038/s41467-024-48953-3
Science. 2024 Jun 07. 384(6700): eadk0850

Determining the ERK-regulated phosphoproteome driving KRAS-mutant cancer.

Jennifer E Klomp, J Nathaniel Diehl, Jeffrey A Klomp, A Cole Edwards, Runying Yang, Alexis J Morales, Khalilah E Taylor, Kristina Drizyte-Miller, Kirsten L Bryant, Antje Schaefer, Jared L Johnson, Emily M Huntsman, Tomer M Yaron, Mariaelena Pierobon, Elisa Baldelli, Alex W Prevatte, Natalie K Barker, Laura E Herring, Emanuel F Petricoin, Lee M Graves, Lewis C Cantley, Adrienne D Cox, Channing J Der, Clint A Stalnecker.

To delineate the mechanisms by which the ERK1 and ERK2 mitogen-activated protein kinases support mutant KRAS-driven cancer growth, we determined the ERK-dependent phosphoproteome in KRAS-mutant pancreatic cancer. We determined that ERK1 and ERK2 share near-identical signaling and transforming outputs and that the KRAS-regulated phosphoproteome is driven nearly completely by ERK. We identified 4666 ERK-dependent phosphosites on 2123 proteins, of which 79 and 66%, respectively, were not previously associated with ERK, substantially expanding the depth and breadth of ERK-dependent phosphorylation events and revealing a considerably more complex function for ERK in cancer. We established that ERK controls a highly dynamic and complex phosphoproteome that converges on cyclin-dependent kinase regulation and RAS homolog guanosine triphosphatase function (RHO GTPase). Our findings establish the most comprehensive molecular portrait and mechanisms by which ERK drives KRAS-dependent pancreatic cancer growth.

DOI: https://doi.org/10.1126/science.adk0850
MedComm (2020). 2024 Jun;5(6): e575

PROteolysis-Targeting Chimeras (PROTACs) in leukemia: overview and future perspectives.

André T S Vicente, Jorge A R Salvador.

  Leukemia is a heterogeneous group of life-threatening malignant disorders of the hematopoietic system. Immunotherapy, radiotherapy, stem cell transplantation, targeted therapy, and chemotherapy are among the approved leukemia treatments. Unfortunately, therapeutic resistance, side effects, relapses, and long-term sequelae occur in a significant proportion of patients and severely compromise the treatment efficacy. The development of novel approaches to improve outcomes is therefore an unmet need. Recently, novel leukemia drug discovery strategies, including targeted protein degradation, have shown potential to advance the field of personalized medicine for leukemia patients. Specifically, PROteolysis-TArgeting Chimeras (PROTACs) are revolutionary compounds that allow the selective degradation of a protein by the ubiquitin-proteasome system. Developed against a wide range of cancer targets, they show promising potential in overcoming many of the drawbacks associated with conventional therapies. Following the exponential growth of antileukemic PROTACs, this article reviews PROTAC-mediated degradation of leukemia-associated targets. Chemical structures, in vitro and in vivo activities, pharmacokinetics, pharmacodynamics, and clinical trials of PROTACs are critically discussed. Furthermore, advantages, challenges, and future perspectives of PROTACs in leukemia are covered, in order to understand the potential that these novel compounds may have as future drugs for leukemia treatment.

Keywords:  PROteolysis‐TArgeting Chimera (PROTAC); bifunctional molecule; degrader; hematological malignancy; leukemia; targeted protein degradation

DOI:  https://doi.org/10.1002/mco2.575
Nat Aging. 2024 Jun 04.

Epigenetic and proteomic signatures associate with clonal hematopoiesis expansion rate.

Taralynn M Mack, Michael A Raddatz, Yash Pershad, Daniel C Nachun, Kent D Taylor, Xiuqing Guo, Alan R Shuldiner, Jeffrey R O'Connell, Eimear E Kenny, Ruth J F Loos, Susan Redline, Brian E Cade, Bruce M Psaty, Joshua C Bis, Jennifer A Brody, Edwin K Silverman, Jeong H Yun, Michael H Cho, Dawn L DeMeo, Daniel Levy, Andrew D Johnson, Rasika A Mathias, Lisa R Yanek, Susan R Heckbert, Nicholas L Smith, Kerri L Wiggins, Laura M Raffield, April P Carson, Jerome I Rotter, Stephen S Rich, Ani W Manichaikul, C Charles Gu, Yii-Der Ida Chen, Wen-Jane Lee, M Benjamin Shoemaker, Dan M Roden, Charles Kooperberg, Paul L Auer, Pinkal Desai, Thomas W Blackwell, Albert V Smith, Alexander P Reiner, Siddhartha Jaiswal, Joshua S Weinstock, Alexander G Bick.

Clonal hematopoiesis of indeterminate potential (CHIP), whereby somatic mutations in hematopoietic stem cells confer a selective advantage and drive clonal expansion, not only correlates with age but also confers increased risk of morbidity and mortality. Here, we leverage genetically predicted traits to identify factors that determine CHIP clonal expansion rate. We used the passenger-approximated clonal expansion rate method to quantify the clonal expansion rate for 4,370 individuals in the National Heart, Lung, and Blood Institute (NHLBI) Trans-Omics for Precision Medicine (TOPMed) cohort and calculated polygenic risk scores for DNA methylation aging, inflammation-related measures and circulating protein levels. Clonal expansion rate was significantly associated with both genetically predicted and measured epigenetic clocks. No associations were identified with inflammation-related lab values or diseases and CHIP expansion rate overall. A proteome-wide search identified predicted circulating levels of myeloid zinc finger 1 and anti-Müllerian hormone as associated with an increased CHIP clonal expansion rate and tissue inhibitor of metalloproteinase 1 and glycine N-methyltransferase as associated with decreased CHIP clonal expansion rate. Together, our findings identify epigenetic and proteomic patterns associated with the rate of hematopoietic clonal expansion.

DOI: https://doi.org/10.1038/s43587-024-00647-7