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1
Heidenreich, W. F. "On the parameters of the clonal expansion model." Radiation and Environmental Biophysics 35, no. 2 (May 1996): 127–29. http://dx.doi.org/10.1007/bf02434036.
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Heidenreich, W. F. "On the parameters of the clonal expansion model." Radiation and Environmental Biophysics 35, no. 2 (May 29, 1996): 127–29. http://dx.doi.org/10.1007/s004110050020.
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Luzzatto, Lucio, and Rosario Notaro. "The “escape” model: a versatile mechanism for clonal expansion." British Journal of Haematology 184, no. 3 (January 24, 2018): 465–66. http://dx.doi.org/10.1111/bjh.15111.
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Shin, Taehoon, Shirley Chen, Stefan Cordes, Yifan Zhou, Byung-Chul Lee, Aisha Aljanahi, So Gun Hong, Robert E. Donahue, Kyung-Rok Yu, and Cynthia E. Dunbar. "Macaque CRISPR/Cas9 Age-Related Clonal Hematopoiesis Model Demonstrates Expansion of TET2-Mutated Clones and Applicability for Testing Mitigation Approaches." Blood 136, Supplement 1 (November 5, 2020): 27–28. http://dx.doi.org/10.1182/blood-2020-140770.
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A series of large-scale genomic studies has reported that clonally expanded hematopoietic cells bearing somatic mutations are increasingly prevalent with age, even in the absence of cytopenias, myelodysplasia, or leukemia. Individuals with acquired somatic mutations at a variant allele frequency (VAF) of at least 2% in genes recurrently mutated in hematologic malignancies not meeting criteria for any known hematologic disorders have been labeled as manifesting "age-related clonal hematopoiesis (ARCH)". Dominant negative or loss-of-function (LOF) mutations in genes encoding for epigenetic modifier enzymes such as DNMT3A, TET2, and ASXL1 are most common in ARCH, and individuals with ARCH are at a greater risk for cardiovascular diseases as well as hematologic malignancies. However, the relationships between these mutations, clonal expansion, and clinical outcomes are not fully elucidated due to difficulties in studying individuals with ARCH longitudinally over time in the absence of an overt clinical abnormality, and extrapolating from murine models that may not closely recapitulate human hematopoietic physiology. Since non-human primates (NHP) have a high similarity in HSPC and marrow properties to humans, and we have identified typical spontaneous ARCH mutations in aged macaques not yet identified in aged mice, we sought to generate a rhesus macaque model of human ARCH utilizing CRISPR/Cas9 technology to investigate clonal behavior and intervention strategies. We delivered a gRNA pool targeting the three most frequently mutated genes in human ARCH with Cas9 in the form of ribonucleoprotein (RNP) into HSPCs obtained from three young adult macaques, targeting a low efficiency, and the edited HSPCs were reinfused into autologous animals following total body irradiation. All macaques engrafted promptly after transplantation and maintained normal blood counts. Up to three years of long-term follow-up revealed reproducible and significant expansion of multiple HSPC clones with heterozygous TET2 LOF mutations, compared to the limited expansion of clones carrying DNMT3A and ASXL1 mutations, reaching a VAF almost 25% with doubling time of 7.5 months in circulating granulocytes of the first macaque (ZL26, Fig 1A). Although there were differences in population doubling rates between individuals, the three macaques shared the general pattern of a gradual but dramatic expansion of TET2-mutated clones, with most of the expanding indels resulting in frameshifts predicted to result in LOF. These data suggest a single mutation in TET2 is sufficient for clonal expansion, and that other intrinsic and/or extrinsic factors can regulate the pace of TET2 clonal expansion. Bone marrow of these macaques exhibited hypercellularity and myeloid-predominant skewing without dysplastic changes compared with macaques of similar age previously transplanted with HSPCs edited at non-ARCH loci. Furthermore, RNA-seq indicated that TET2-disrupted myeloid colony-forming units (CFUs) and mature cells exhibited a distinct hyperinflammatory gene expression profile. Indeed, CD14+CD163+ macrophages purified from all three ARCH macaques exhibited hyperinflammatory function, with upregulated NLRP3 inflammasome activity and increased IL-6 signaling. We hypothesized that interrupting the vicious cycle of clonal expansion driven by and driving inflammation could halt the expansion of TET2-mutated clones. To address this, we treated the animal with the fastest TET2-mutant clonal expansion (ZH63) with tocilizumab, an antibody blocking IL-6 signaling, starting 13 months after transplantation and continuing for 4 months. The TET2 mutated allele frequency in granulocytes declined by 30% by the end of the treatment and began to increase again after withdrawal (Fig 1B), suggesting that interruption of the IL-6 axis removes the selective advantage of mutant HSPCs and this repressive effect is specific to the TET2-mutant genotype. In summary, our CRISPR/Cas9-engineered rhesus macaque ARCH model recapitulates human ARCH and uncovers the impact of TET2 LOF on hematopoiesis and inflammation, as well as demonstrates the suppressive effect of IL-6 axis blockade in TET2-mutant clonal expansions. This robust NHP model will be further utilized for examining the pathophysiology of ARCH and testing of potential therapeutic interventions. Disclosures Dunbar: Novartis: Research Funding.
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Murray, John M. "Dynamics of latent HIV under clonal expansion." PLOS Pathogens 17, no. 12 (December 20, 2021): e1010165. http://dx.doi.org/10.1371/journal.ppat.1010165.
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The HIV latent reservoir exhibits slow decay on antiretroviral therapy (ART), impacted by homeostatic proliferation and activation. How these processes contribute to the total dynamic while also producing the observed profile of sampled latent clone sizes is unclear. An agent-based model was developed that tracks individual latent clones, incorporating homeostatic proliferation of cells and activation of clones. The model was calibrated to produce observed latent reservoir dynamics as well as observed clonal size profiles. Simulations were compared to previously published latent HIV integration data from 5 adults and 3 children. The model simulations reproduced reservoir dynamics as well as generating residual plasma viremia levels (pVL) consistent with observations on ART. Over 382 Latin Hypercube Sample simulations, the median latent reservoir grew by only 0.3 log10 over the 10 years prior to ART initiation, after which time it decreased with a half-life of 15 years, despite number of clones decreasing at a faster rate. Activation produced a maximum size of genetically intact clones of around one million cells. The individual simulation that best reproduced the sampled clone profile, produced a reservoir that decayed with a 13.9 year half-life and where pVL, produced mainly from proliferation, decayed with a half-life of 10.8 years. These slow decay rates were achieved with mean cell life-spans of only 14.2 months, due to expansion of the reservoir through proliferation and activation. Although the reservoir decayed on ART, a number of clones increased in size more than 4,000-fold. While small sampled clones may have expanded through proliferation, the large sizes exclusively arose from activation. Simulations where homeostatic proliferation contributed more to pVL than activation, produced pVL that was less variable over time and exhibited fewer viral blips. While homeostatic proliferation adds to the latent reservoir, activation can both add and remove latent cells. Latent activation can produce large clones, where these may have been seeded much earlier than when first sampled. Elimination of the reservoir is complicated by expanding clones whose dynamic differ considerably to that of the entire reservoir.
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van der Heijden, Maartje, Daniël M. Miedema, Bartlomiej Waclaw, Veronique L. Veenstra, Maria C. Lecca, Lisanne E. Nijman, Erik van Dijk, et al. "Spatiotemporal regulation of clonogenicity in colorectal cancer xenografts." Proceedings of the National Academy of Sciences 116, no. 13 (March 8, 2019): 6140–45. http://dx.doi.org/10.1073/pnas.1813417116.
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Cancer evolution is predominantly studied by focusing on differences in the genetic characteristics of malignant cells within tumors. However, the spatiotemporal dynamics of clonal outgrowth that underlie evolutionary trajectories remain largely unresolved. Here, we sought to unravel the clonal dynamics of colorectal cancer (CRC) expansion in space and time by using a color-based clonal tracing method. This method involves lentiviral red-green-blue (RGB) marking of cell populations, which enabled us to track individual cells and their clonal outgrowth during tumor initiation and growth in a xenograft model. We found that clonal expansion largely depends on the location of a clone, as small clones reside in the center and large clones mostly drive tumor growth at the border. These dynamics are recapitulated in a computational model, which confirms that the clone position within a tumor rather than cell-intrinsic features, is crucial for clonal outgrowth. We also found that no significant clonal loss occurs during tumor growth and clonal dispersal is limited in most models. Our results imply that, in addition to molecular features of clones such as (epi-)genetic differences between cells, clone location and the geometry of tumor growth are crucial for clonal expansion. Our findings suggest that either microenvironmental signals on the tumor border or differences in physical properties within the tumor, are major contributors to explain heterogeneous clonal expansion. Thus, this study provides further insights into the dynamics of solid tumor growth and progression, as well as the origins of tumor cell heterogeneity in a relevant model system.
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Fleischman, Angela G., Karl J. Aichberger, Samuel B. Luty, Thomas G. Bumm, Curtis L. Petersen, Shirin Doratotaj, Kavin B. Vasudevan, et al. "TNFα facilitates clonal expansion of JAK2V617F positive cells in myeloproliferative neoplasms." Blood 118, no. 24 (December 8, 2011): 6392–98. http://dx.doi.org/10.1182/blood-2011-04-348144.
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AbstractProinflammatory cytokines such as TNFα are elevated in patients with myeloproliferative neoplasms (MPN), but their contribution to disease pathogenesis is unknown. Here we reveal a central role for TNFα in promoting clonal dominance of JAK2V617F expressing cells in MPN. We show that JAK2V617F kinase regulates TNFα expression in cell lines and primary MPN cells and TNFα expression is correlated with JAK2V617F allele burden. In clonogenic assays, normal controls show reduced colony formation in the presence of TNFα while colony formation by JAK2V617F-positive progenitor cells is resistant or stimulated by exposure to TNFα. Ectopic JAK2V617F expression confers TNFα resistance to normal murine progenitor cells and overcomes inherent TNFα hypersensitivity of Fanconi anemia complementation group C deficient progenitors. Lastly, absence of TNFα limits clonal expansion and attenuates disease in a murine model of JAK2V617F-positive MPN. Altogether our data are consistent with a model where JAK2V617F promotes clonal selection by conferring TNFα resistance to a preneoplastic TNFα sensitive cell, while simultaneously generating a TNFα-rich environment. Mutations that confer resistance to environmental stem cell stressors are a recognized mechanism of clonal selection and leukemogenesis in bone marrow failure syndromes and our data suggest that this mechanism is also critical to clonal selection in MPN.
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Chen, Yong, Lingyun Shao, Zahida Ali, Jiye Cai, and Zheng W. Chen. "NSOM/QD-based nanoscale immunofluorescence imaging of antigen-specific T-cell receptor responses during an in vivo clonal Vγ2Vδ2 T-cell expansion." Blood 111, no. 8 (April 15, 2008): 4220–32. http://dx.doi.org/10.1182/blood-2007-07-101691.
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Abstract Nanoscale imaging of an in vivo antigen-specific T-cell immune response has not been reported. Here, the combined near-field scanning optical microscopy– and fluorescent quantum dot–based nanotechnology was used to perform immunofluorescence imaging of antigen-specific T-cell receptor (TCR) response in an in vivo model of clonal T-cell expansion. The near-field scanning optical microscopy/quantum dot system provided a best-optical-resolution (<50 nm) nano-scale imaging of Vγ2Vδ2 TCR on the membrane of nonstimulated Vγ2Vδ2 T cells. Before Ag-induced clonal expansion, these nonstimulating Vγ2Vδ2 TCRs appeared to be distributed differently from their αβ TCR counterparts on the cell surface. Surprisingly, Vγ2Vδ2 TCR nanoclusters not only were formed but also sustained on the membrane during an in vivo clonal expansion of Vγ2Vδ2 T cells after phosphoantigen treatment or phosphoantigen plus mycobacterial infection. The TCR nanoclusters could array to form nanodomains or microdomains on the membrane of clonally expanded Vγ2Vδ2 T cells. Interestingly, expanded Vγ2Vδ2 T cells bearing TCR nanoclusters or nanodomains were able to rerecognize phosphoantigen and to exert better effector function. These studies provided nanoscale insight into the in vivo T-cell immune response.
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Newgreen, Donald F., Dongcheng Zhang, Bevan L. Cheeseman, Benjamin J. Binder, and Kerry A. Landman. "Differential Clonal Expansion in an Invading Cell Population: Clonal Advantage or Dumb Luck?" Cells Tissues Organs 203, no. 2 (2017): 105–13. http://dx.doi.org/10.1159/000452793.
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In neoplastic cell growth, clones and subclones are variable both in size and mutational spectrum. The largest of these clones are believed to represent those cells with mutations that make them the most “fit,” in a Darwinian sense, for expansion in their microenvironment. Thus, the degree of quantitative clonal expansion is regarded as being determined by innate qualitative differences between the cells that originate each clone. Here, using a combination of mathematical modelling and clonal labelling experiments applied to the developmental model system of the forming enteric nervous system, we describe how cells which are qualitatively identical may consistently produce clones of dramatically different sizes: most clones are very small while a few clones we term “superstars” contribute most of the cells to the final population. The basis of this is minor stochastic variations (“luck”) in the timing and direction of movement and proliferation of individual cells, which builds a local advantage for daughter cells that is cumulative. This has potentially important consequences. In cancers, especially before strongly selective cytotoxic therapy, the assumption that the largest clones must be the cells with deterministic proliferative ability may not always hold true. In development, the gradual loss of clonal diversity as “superstars” take over the population may erode the resilience of the system to somatic mutations, which may have occurred early in clonal growth.
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Fu, Xiao, Yue Zhao, Jose I. Lopez, Andrew Rowan, Lewis Au, Annika Fendler, Steve Hazell, et al. "Spatial patterns of tumour growth impact clonal diversification in a computational model and the TRACERx Renal study." Nature Ecology & Evolution 6, no. 1 (December 23, 2021): 88–102. http://dx.doi.org/10.1038/s41559-021-01586-x.
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AbstractGenetic intra-tumour heterogeneity fuels clonal evolution, but our understanding of clinically relevant clonal dynamics remain limited. We investigated spatial and temporal features of clonal diversification in clear cell renal cell carcinoma through a combination of modelling and real tumour analysis. We observe that the mode of tumour growth, surface or volume, impacts the extent of subclonal diversification, enabling interpretation of clonal diversity in patient tumours. Specific patterns of proliferation and necrosis explain clonal expansion and emergence of parallel evolution and microdiversity in tumours. In silico time-course studies reveal the appearance of budding structures before detectable subclonal diversification. Intriguingly, we observe radiological evidence of budding structures in early-stage clear cell renal cell carcinoma, indicating that future clonal evolution may be predictable from imaging. Our findings offer a window into the temporal and spatial features of clinically relevant clonal evolution.
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Caiado, Francisco, Larisa V. Kovtonyuk, and Markus G. Manz. "Clonal Expansion of Tet2 +/- hematopoiesis Is Driven By Inflamm-Ageing Associated IL-1 Increase in Mice." Blood 138, Supplement 1 (November 5, 2021): 1086. http://dx.doi.org/10.1182/blood-2021-146815.
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Abstract Clonal Hematopoiesis of Indeterminate Potential (CHIP) is defined as the presence of an expanded somatic blood cell clone carrying a mutation in genes that are drivers of hematologic malignancy including DNMT3A, TET2, ASXL1, TP53, JAK2, and SF3B1, at a variant allele frequency (VAF) of at least 2% in the absence of other hematological abnormalities. CHIP has a prevalence of about 10% in the 70-80 year old population, further increases with ageing and associates with an increased risk of hematological malignancies, cardiovascular disease and all-cause mortality (Genovese et al. NEJM 2014; Jaiswal, S. et al. NEJM 2014). Recent studies indicate that higher pre-malignant clonal size and mutational burden increase the chances of malignant transformation in individuals carrying CHIP (Desai, P. Nat. Med. et al., 2018; Abelson, S. et al. Nature, 2018). While age is the best predictor of CHIP development and correlates directly with pre-malignant clonal size, the specific cellular-extrinsic factors promoting CHIP clonal expansion in the context of physiological aging are still unclear. We hypothesized that ageing associated low-grade inflammation (termed "inflamm-ageing") is a driver of CHIP clonal expansion. We used standard bone marrow (BM) chimera models and developed a novel, irradiation independent, hematopoietic specific and tamoxifen inducible genetic mosaicism mouse model of Tet2 +/- driven CHIP (HSC-Scl-Cre-ERT; Tet2+/flox; R26 +/floxstop-EGFP triple transgenic mice) to determine the contribution of inflamm-aging factors to Tet2 +/- hematopoieticclonal expansion. Using these complementary models, we observe that peripheral Tet2 +/- clonal expansion rates increase with age (evident in erythroid, myeloid, lymphoid B and T lineages), which is paralleled by a significant expansion of Tet2 +/- hematopoietic stem and progenitor cell (HSPCs) populations in aged mice (12-14 months old). Importantly, Tet2 +/- clonal expansion associates with increased levels of inflammatory cytokine IL-1 in aged mice, which derives partially from Tet2 +/- mutant mature hematopoietic cells. To test the contribution of IL-1 to Tet2 +/- clonal expansion, we administered IL-1 (0.5ug/day for 14 days) to young CHIP carrying mice (2-4 months of age) and observed an IL-1R1-dependent expansion of Tet2 +/- hematopoietic mature lineages and HSPCs. Dissection of the cellular mechanisms operating downstream of IL-1/IL-1R1 revealed that Tet2 +/- clonal expansion results from increased multilineage differentiation and associates with increased HSPC cell-cycle progression (while not depending on IL-1-mediated effects on HSPC viability). Moreover, Tet2 +/- HSPCs show a higher in vitro and in vivo repopulation capacity in response to prolonged IL-1 exposure compared to their WT counterparts. Finally, to directly test the contribution of IL-1 to drive Tet2 +/- clonal expansion in the context of physiological aging, we set up genetic (BM chimeras using donor BM from Tet2 +/-; Il-1r1-/- compound mutants) or pharmacological inhibited IL-1 signaling (Anakinra, hIL-1ra) during mouse ageing. Strikingly, both approaches prevented ageing-dependent Tet2 +/- clonal expansion, thus confirming IL-1 as key "inflamm-ageing" driver of Tet2 +/- clonal expansion. Overall, our data provide proof-of-concept that IL-1 production derived from aged BM cells is a relevant and targetable driver of Tet2 +/- clonal expansion in aged mice. Disclosures Manz: CDR-Life Inc: Consultancy, Current holder of stock options in a privately-held company; University of Zurich: Patents & Royalties: CD117xCD3 TEA.
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Kaiser, J. C., and W. F. Heidenreich. "Comparing regression methods for the two-stage clonal expansion model of carcinogenesis." Statistics in Medicine 23, no. 21 (2004): 3333–50. http://dx.doi.org/10.1002/sim.1620.
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Yamamoto, Kimiyo N., Lin L. Liu, Akira Nakamura, Hiroshi Haeno, and Franziska Michor. "Stochastic Evolution of Pancreatic Cancer Metastases During Logistic Clonal Expansion." JCO Clinical Cancer Informatics, no. 3 (December 2019): 1–11. http://dx.doi.org/10.1200/cci.18.00079.
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Despite recent progress in diagnostic and multimodal treatment approaches, most cancer deaths are still caused by metastatic spread and the subsequent growth of tumor cells in sites distant from the primary organ. So far, few quantitative studies are available that allow for the estimation of metastatic parameters and the evaluation of alternative treatment strategies. Most computational studies have focused on situations in which the tumor cell population expands exponentially over time; however, tumors may eventually be subject to resource and space limitations so that their growth patterns deviate from exponential growth to adhere to density-dependent growth models. In this study, we developed a stochastic evolutionary model of cancer progression that considers alterations in metastasis-related genes and intercellular growth competition leading to density effects described by logistic growth. Using this stochastic model, we derived analytical approximations for the time between the initiation of tumorigenesis and diagnosis, the expected number of metastatic sites, the total number of metastatic cells, the size of the primary tumor, and survival. Furthermore, we investigated the effects of drug administration and surgical resection on these quantities and predicted outcomes for different treatment regimens. Parameter values used in the analysis were estimated from data obtained from a pancreatic cancer rapid autopsy program. Our theoretical approach allows for flexible modeling of metastatic progression dynamics.
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An, Ningfei, Saira Khan, Molly K. Imgruet, Sandeep K. Gurbuxani, Stephanie N. Konecki, Michael R. Burgess, and Megan E. McNerney. "Gene dosage effect of CUX1 in a murine model disrupts HSC homeostasis and controls the severity and mortality of MDS." Blood 131, no. 24 (June 14, 2018): 2682–97. http://dx.doi.org/10.1182/blood-2017-10-810028.
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Key Points CUX1 deficiency leads to transient clonal expansion followed by HSC depletion, anemia, and trilineage dysplasia. CUX1 transcriptionally regulates HSC quiescence, proliferation, and lineage specification.
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Castrén, Olli. "IMPLICATIONS OF A TWO-STAGE CLONAL EXPANSION MODEL TO INDOOR RADON RISK ASSESSMENT." Health Physics 76, no. 4 (April 1999): 393–97. http://dx.doi.org/10.1097/00004032-199904000-00007.
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M Zielinski, R L Kodell, D Krewski, J. "Interaction between two carcinogens in the two-stage clonal expansion model of carcinogenesis." Journal of Epidemiology and Biostatistics 6, no. 2 (March 1, 2001): 219–28. http://dx.doi.org/10.1080/135952201753172999.
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Heidenreich, Wolfgang F., E. Georg Luebeck, and Suresh H. Moolgavkar. "Some Properties of the Hazard Function of the Two-Mutation Clonal Expansion Model." Risk Analysis 17, no. 3 (June 1997): 391–99. http://dx.doi.org/10.1111/j.1539-6924.1997.tb00878.x.
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Tan, E., N. Warren, A. Darnton, and J. Hodgson. "Modelling mesothelioma mortality in Great Britain using the two-stage clonal expansion model." Occupational and Environmental Medicine 68, Suppl_1 (September 1, 2011): A60. http://dx.doi.org/10.1136/oemed-2011-100382.194.
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Kaiser, J. C., and W. F. Heidenreich. "Identifying dose dependences of the two-stage clonal expansion model with simulated cohorts." Journal of Radiological Protection 22, no. 3A (September 1, 2002): A57—A60. http://dx.doi.org/10.1088/0952-4746/22/3a/310.
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Hageb, Ali, Torsten Thalheim, Kalpana J. Nattamai, Bettina Möhrle, Mehmet Saçma, Vadim Sakk, Lars Thielecke, et al. "Reduced adhesion of aged intestinal stem cells contributes to an accelerated clonal drift." Life Science Alliance 5, no. 8 (April 29, 2022): e202201408. http://dx.doi.org/10.26508/lsa.202201408.
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Upon aging, the function of the intestinal epithelium declines with a concomitant increase in aging-related diseases. ISCs play an important role in this process. It is known that ISC clonal dynamics follow a neutral drift model. However, it is not clear whether the drift model is still valid in aged ISCs. Tracking of clonal dynamics by clonal tracing revealed that aged crypts drift into monoclonality substantially faster than young ones. However, ISC tracing experiments, in vivo and ex vivo, implied a similar clonal expansion ability of both young and aged ISCs. Single-cell RNA sequencing for 1,920 high Lgr5 ISCs from young and aged mice revealed increased heterogeneity among subgroups of aged ISCs. Genes associated with cell adhesion were down-regulated in aged ISCs. ISCs of aged mice indeed show weaker adhesion to the matrix. Simulations applying a single cell–based model of the small intestinal crypt demonstrated an accelerated clonal drift at reduced adhesion strength, implying a central role for reduced adhesion for affecting clonal dynamics upon aging.
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Pélissier, Aurélien, Youcef Akrout, Katharina Jahn , Jack Kuipers , Ulf Klein , Niko Beerenwinkel, and María Rodríguez Martínez. "Computational Model Reveals a Stochastic Mechanism behind Germinal Center Clonal Bursts." Cells 9, no. 6 (June 10, 2020): 1448. http://dx.doi.org/10.3390/cells9061448.
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Germinal centers (GCs) are specialized compartments within the secondary lymphoid organs where B cells proliferate, differentiate, and mutate their antibody genes in response to the presence of foreign antigens. Through the GC lifespan, interclonal competition between B cells leads to increased affinity of the B cell receptors for antigens accompanied by a loss of clonal diversity, although the mechanisms underlying clonal dynamics are not completely understood. We present here a multi-scale quantitative model of the GC reaction that integrates an intracellular component, accounting for the genetic events that shape B cell differentiation, and an extracellular stochastic component, which accounts for the random cellular interactions within the GC. In addition, B cell receptors are represented as sequences of nucleotides that mature and diversify through somatic hypermutations. We exploit extensive experimental characterizations of the GC dynamics to parameterize our model, and visualize affinity maturation by means of evolutionary phylogenetic trees. Our explicit modeling of B cell maturation enables us to characterise the evolutionary processes and competition at the heart of the GC dynamics, and explains the emergence of clonal dominance as a result of initially small stochastic advantages in the affinity to antigen. Interestingly, a subset of the GC undergoes massive expansion of higher-affinity B cell variants (clonal bursts), leading to a loss of clonal diversity at a significantly faster rate than in GCs that do not exhibit clonal dominance. Our work contributes towards an in silico vaccine design, and has implications for the better understanding of the mechanisms underlying autoimmune disease and GC-derived lymphomas.
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Wu, Chuanfeng, Jan K. Davidson-Moncada, Rong Lu, Brian Li, Allen E. Krouse, Mark E. Metzger, Robert E. Donahue, Irving L. Weissman, Richard Childs, and Cynthia E. Dunbar. "The Unique Ontogeny Of Natural Killer Cells As Revealed By Genetic Barcoding In The Nonhuman Primate Model." Blood 122, no. 21 (November 15, 2013): 15. http://dx.doi.org/10.1182/blood.v122.21.15.15.
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Abstract Natural killer (NK) cells are cytotoxic leukocytes defined in humans and rhesus macaques by the expression of CD56 and/or CD16, and the absence of T, B, and myeloid markers. Functionally, they are defined by lysis of tumor targets in a major histocompatibility complex (MHC)–unrestricted fashion, and production of cytokines critical to innate immunity. Ex vivo expanded NK cells are being developed as adoptive transfer therapeutics for a variety of malignancies. However, the ontogeny and hematopoietic lineage relationships of human NK cells have been difficult to study, given differences in phenotype and function between human and murine NK cells, poor NK engraftment and development in human xenografts, and restricted development of NK cells from hematopoietic stem and progenitor cells (HSPC) in vitro. In addition, several phenotypic subsets of NK cells have been described, and their relationships and relative locations within the hematopoietic hierarchy are supported by limited in vivo data. We “barcoded” individual CD34+ HSPC from 3 macaques using lentiviral vectors carrying highly diverse oligonucleotides, allowing precise quantitation of clonal contributions via low cycle PCR amplification followed by high-throughput sequencing and computational analysis (Lu et al, Nat Biotech). We quantitated barcode levels, and thus individual HSPC clonal contributions, over time and between lineages, following TBI and infusion of autologous barcoded CD34+ cells. Reconstitution of the NK compartment was clonally distinct from T, B and myeloid (My) lineages. Following reconstitution of NK, T, B and My from uni-lineage progenitors at 1-2 months(m), by 3-4 m, as expected from murine and in vitro models, multi-lineage clones began to contribute and dominate, first B/My, then B/T/My. However, NK cells remained clonally distinct through 9 m, despite overall clonal diversity and marking levels in NK lineage similar to B/T and My. Even the most abundant clones in the NK lineage were not found contributing in any significant way to B/T or My lineages. Shared B/T/My/NK clones finally began to contribute more extensively at 9-14 m. We fractionated peripheral blood NK cells into 3 NK subsets and compared them to overall PB and lymph node NK cells at 4.5-6.5 m. The majority (75-90%) of PB rhesus NK cells are found in the CD16+/CD56- PB subset (corresponding to human CD16+/CD56dim cytotoxic NK), and this subset accounted for the clonal pattern in overall NK cells (Pearson r=0.98 vs overall NK cells), and the disparity from B/T/My, with r values all <0.17 for CD16+/CD56- vs B/T or My lineages at the same time point. The minor PB (but dominant lymph node) cytokine-producing CD16-/CD56+ or CD16+/CD56+ NK subsets, previously hypothesized to be precursors for CD16+/CD56- NK cells, had clonal patterns that were more closely correlated with T/B/My lineages (r=0.37-0.62) than the CD16+/CD56- cells. The clonal correlations between the putative precursor CD16-/CD56+ cells and mature cytotoxic CD16+/CD56- cells was very low, only r=0.08. Furthermore, we expanded purified NK cells from barcoded macaques in vitro in the presence of IL-2 and irradiated EBV-LCL cells and assessed NK phenotype, function, and clonal diversity over time. 40-90X expansion was achieved by 15 days and was highly polyclonal, with 90% of the starting number of barcodes still present at the end of expansion. Clonal contribution levels between pre and post-expanded NK were highly correlated (r=0.73). CD16+/CD56+ cells became more dominant in post-exp NK cells, in contrast to the majority CD16+CD56- pre-exp. The clonal contributions in the post-exp CD16+/CD56- and CD16+/CD56+ cells correlated with each other (r=0.66), and with the starting CD16+/CD56- cells (r=0.75), but not with the starting CD16-/CD56+ (r=0.15) nor the post-exp CD16-/CD56+ cells (r=0.18). Our in vivo and in vitro results call into question the hypothesis that CD16-/CD56+ NK cells are precursors of circulating cytotoxic CD16+/CD56- NK cells, as does the complete deficiency of CD16-/CD56+ but not CD16+/CD56dim cells in GATA2 mutant patients. Our results also reveal that the dominant blood rhesus NK cell population has a distinct ontogeny in macaques, and thus potentially in humans. NK cells expanded ex vivo using EBV-LCL cells are polyclonal, and barcoding allows dissection of events during expansion, and will permit tracking studies of expanded cells following adoptive transfer. Disclosures: No relevant conflicts of interest to declare.
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Eshetu, Amare, and Ya-Chi Ho. "A multidimensional HIV-1 persistence model for clonal expansion and viral rebound in vitro." Cell Reports Methods 1, no. 8 (December 2021): 100134. http://dx.doi.org/10.1016/j.crmeth.2021.100134.
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Heidenreich, Wolfgang F., and Rudolf Hoogenveen. "Limits of Applicability for the Deterministic Approximation of the Two-Step Clonal Expansion Model." Risk Analysis 21, no. 1 (February 2001): 103–6. http://dx.doi.org/10.1111/0272-4332.211093.
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Geisler, Iris, and Annette Kopp-Schneider. "A model for hepatocarcinogenesis with clonal expansion of three successive phenotypes of preneoplastic cells." Mathematical Biosciences 168, no. 2 (December 2000): 167–85. http://dx.doi.org/10.1016/s0025-5564(00)00044-4.
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Kodell, Ralph L., Daniel Krewski, and Jan M. Zielinski. "Additive and Multiplicative Relative Risk in the Two-Stage Clonal Expansion Model of Carcinogenesis." Risk Analysis 11, no. 3 (September 1991): 483–90. http://dx.doi.org/10.1111/j.1539-6924.1991.tb00633.x.
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Wang, Qun, Yen-Yu Lin, Baojun Zhang, Jianxuan Wu, Sumedha Roy, Jeremy J. Ratiu, Yanping Xu, et al. "A mosaic analysis system with Cre or Tomato expression in the mouse." Proceedings of the National Academy of Sciences 117, no. 45 (October 26, 2020): 28212–20. http://dx.doi.org/10.1073/pnas.2014308117.
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Somatic mutations are major genetic contributors to cancers and many other age-related diseases. Many disease-causing somatic mutations can initiate clonal growth prior to the appearance of any disease symptoms, yet experimental models that can be used to examine clonal abnormalities are limited. We describe a mosaic analysis system with Cre or Tomato (MASCOT) for tracking mutant cells and demonstrate its utility for modeling clonal hematopoiesis. MASCOT can be induced to constitutively express either Cre-GFP or Tomato for lineage tracing of a mutant and a reference group of cells simultaneously. We conducted mosaic analysis to assess functions of theId3and/orTet2gene in hematopoietic cell development and clonal hematopoiesis. Using Tomato-positive cells as a reference population, we demonstrated the high sensitivity of this system for detecting cell-intrinsic phenotypes during short-term or long-term tracking of hematopoietic cells. Long-term tracking ofTet2mutant orTet2/Id3double-mutant cells in our MASCOT model revealed a dynamic shift from myeloid expansion to lymphoid expansion and subsequent development of lymphoma. This work demonstrates the utility of the MASCOT method in mosaic analysis of single or combined mutations, making the system suitable for modeling somatic mutations identified in humans.
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Bamezai, Anil, Daniel Schieffer, and Sanya Naware. "Lipid raft-based membrane order is important for antigen-specific clonal expansion of CD4+ T cells (IRC2P.451)." Journal of Immunology 192, no. 1_Supplement (May 1, 2014): 58.8. http://dx.doi.org/10.4049/jimmunol.192.supp.58.8.
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Abstract Lipid rafts contribute to the plasma membrane order and to its spatial asymmetry. Lipid rafts are cholesterol and saturated lipid-rich, nanometer sized membrane domains that are hypothesized to play an important role in spatiotemporal regulation of cell signaling. The raft nanodomains on the surface of CD4+ T lymphocytes coalesce during their interaction with antigen presenting cells (APCs). Sensing of foreign antigen by the antigen receptor on CD4+ T cells occurs during these cell-cell interactions. In response to foreign antigen the CD4+ T cells proliferate, allowing the expansion of few antigen-specific primary CD4+ T cell clones. Proliferating CD4+ T cells undergo differentiation into appropriate effectors tailored to mount an effective immunity to the pathogen. To investigate the role of lipid raft-based membrane order in the clonal expansion phase of primary CD4+ T cells, we have disrupted membrane order by incorporating an oxysterol, 7-ketocholesterol (7-KC) into the plasma membrane of primary CD4+ T cells expressing a T cell receptor specific to chicken ovalbumin323-339 peptide sequence and tested their antigen-specific response. We report that 7-KC, at appropriate concentrations, significantly diminishes the c-Ovalbumin323-339 peptide-specific clonal expansion of primary CD4+ T cells. Our findings suggest that lipid raft-based membrane order and membrane asymmetry is important for clonal expansion of CD4+ T cells in response to a model antigen.
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Eden, Christopher O., David K. Edwards, Christopher A. Eide, Elie Traer, Jeffrey W. Tyner, Shannon K. McWeeney, and Anupriya Agarwal. "Cytokine-Mediated Inflammatory Pathways Promote Clonal Evolution and Disease Progression in Acute Myeloid Leukemia." Blood 128, no. 22 (December 2, 2016): 1688. http://dx.doi.org/10.1182/blood.v128.22.1688.1688.
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Abstract Background: Inflammatory cytokines secreted in the bone marrow microenvironment play important roles in modulating cell survival, proliferation, differentiation, and immune responses in cancer. Perhaps not surprisingly, there is also an association between chronic inflammation and tumor progression. We recently used an ex vivo functional screen of 94 cytokines to show that the pro-inflammatory cytokines IL1α and IL1β promoted the expansion of AML progenitors in 70% (40/60) of primary samples. We therefore hypothesized that inflammatory cytokines are crucial to clonal expansion and disease progression in AML and that therapeutic targeting of these pathways may circumvent disease heterogeneity. Here we provide in vitro and in vivo evidence that IL1-mediated signaling elicits profound expansion of leukemia progenitors in AML patients harboring various genetic mutations and promotes in vivo clonal expansion and disease progression in a murine AML model. Further, these effects are reversed by targeting IL1 signaling. Methods: We validated the role of IL1 signaling using a shRNA approach and in murine competitive repopulation and bone marrow transplantation models. We evaluated the influence of these cytokines on inflammatory markers using immunoblotting, flow cytometry, and Luminex assays, and assessed strategies to target these pathways using small-molecule inhibitors. Results: IL1 stimulation promoted a 3- to 20-fold increase in growth, survival, and clonogenic potential of AML CD34+ cells, while paradoxically suppressing growth of healthy CD34+ cells. To identify the influence of IL1 on in vivo clonal expansion of healthy and leukemic progenitors simultaneously, we established an in vivo murine competitive repopulation study utilizing TET2-null mice. IL1 treatment promoted clonal expansion of TET2-null myeloid cells over wild-type cells during 6 weeks of IL1β treatment. Consistent with this, both flow cytometry analysis and blood differential counts showed an increased percentage of granulocytes and reduced percentage of lymphocytes in IL1-treated mice. In this model, TET2-null monocytes have greater expression of IL1 than wild-type cells, suggesting IL1 promotes clonal growth of TET2-mutated early leukemic progenitors. Similarly, IL1β and IL1 receptors (IL1R1 and IL1RAP) were overexpressed in IL1-sensitive AML bone marrow and peripheral blood samples compared to nonsensitive AML samples and normal samples. Intracellular FACS showed that the majority of IL1β was secreted by monocytes and to some extent by myeloid progenitors. Accordingly, IL1-sensitive AML samples exhibited trends towards monocytic and myelomonocytic clinical features. Reduced survival of AML cells after monocyte depletion was rescued by IL1 treatment, suggesting that IL1 mediates paracrine regulation of AML cell growth. Silencing of the IL1 receptor, IL1R1, reduced the clonogenic potential of AML primary samples and oncogene (AML1-ETO9a, NRASG12D, and MLL-ENL)-transduced mouse bone marrow. In a murine bone marrow transplantation model, recipients of IL1R1-/- marrow transduced with AML1-ETO9a/NRASG12D survived significantly longer than did recipients of wild-type marrow. We also found that IL1β increased phosphorylation of p38MAPK and MK2, as well as secretion of multiple downstream inflammatory cytokines (IL6, IL8, MCP1, MIP1α, and MIP1β) from CD34+ progenitors, in IL1-sensitive AML samples compared to IL1-nonsensitive progenitors. Conversely, treating AML cells with p38MAPK inhibitors such as doramapimod andralimetinib reduced the growth of AML cells by decreasing p38MAPK and MK2 phosphorylation and reducing secretion of inflammatory cytokines from AML progenitors. Clinical and demographic analyses suggest that AML patients are dependent on IL1 signaling irrespective of mutation status and clinical features. Targeting this unifying mechanism of IL1-mediated clonal expansion may thus have application across heterogeneous AML subtypes. Conclusion: We demonstrate that IL1 promotes in vitro and in vivo clonal expansion of leukemic cells and promotes disease progression in AML. As IL1 signaling is active across heterogeneous disease subtypes, AML patients may therefore benefit from drugs targeting IL1/p38MAPK signaling because of their potential to inhibit AML while enhancing normal hematopoiesis, a significant clinical advantage over traditional chemotherapy. Disclosures Agarwal: CTI BioPharma Corp: Research Funding.
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Murakami, Yoshiko, Norimitsu Inoue, Tsutomu Shichishima, Hideyoshi Noji, Jun-Ichi Nishimura, Yuzuru Kanakura, and Taroh Kinoshita. "Expression of HMGA2 In Blood Cells From Patients with Paroxysmal Nocturnal Hemoglobinuria." Blood 116, no. 21 (November 19, 2010): 4242. http://dx.doi.org/10.1182/blood.v116.21.4242.4242.
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Abstract Abstract 4242 Paroxysmal nocturnal hemoglobinuria (PNH) is caused by a somatic mutation of PIG-A gene in one or few hematopoietic stem cells and subsequent clonal expansion of mutant stem cells. It is known that PIG-A mutation is insufficient to account for the clonal expansion required for clinical manifestation of PNH. We proposed a 3-step model of PNH pathogenesis. Step 1 involves the generation of a GPI-deficient hematopoietic stem cell by somatic mutation of PIG-A gene. Step 2 involves the immunological selection of GPI-deficient hematopoietic stem cells. Based on the close association of PNH with aplastic anemia, it has been suggested that the selection pressure is immune mediated. However, in spite that over 60% of patients with aplastic anemia have subclinical population of GPI-deficient hematopoietic cells at diagnosis, only 10% develop clinical PNH, suggesting that steps-1 and 2 are insufficient to cause PNH. Under immune mediated selection pressure, GPI-deficient cells not only survive, but also must proliferate much more frequently than usual to compensate for anemia. This elevated proliferation rate may increase a chance that additional mutations are acquired, in turn leads to step 3. Step 3 involves a second somatic mutation that bestows on PIG-A mutant stem cell a proliferative phenotype. According to this hypothesis, we searched for the candidate gene for step 3. We reported 2 patients with PNH whose PIG-A mutant cells had an acquired rearrangement of chromosome12, generating the break within the 3′ untranslated region in HMGA2. This gene encodes an architectural transcription factor which is deregulated in many benign mesenchymal tumors (Blood. 2006 vol.108 no.13, p4232). Based on these, we consider HMGA2 as a candidate gene, ectopic expression of which causes proliferation of PIG-A mutant cells. We reported that the expression of HMGA2 in peripheral blood from PNH patients was significantly higher than that from normal volunteers (relative mRNA expression, 4.8±2.4 vs 1.3±0.3, p<0.05) but this was not the case in the bone marrow. We investigated whether over expression of HMGA2 really causes the clonal expansion using the mouse model. We transduced the mouse bone marrow cells with retrovirus vectors, pMYs-HMGA2-IRES-EGFP or pMY-IRES-EGFP as a control, and transplanted them into lethally irradiated mice. The percentage of HMGA2 expressing cells in peripheral blood cells of each lineage from transplanted mice gradually increased during 4-months' observation, suggesting that over expression of HMGA2 caused clonal expansion of multipotent hematopoietic cells. This result is consistent with a recent report on the gene therapy of beta-thalathemia that clonal expansion of rescued hematopoietic cells occurred due to lentivaral insertion into and ectopic expression of HMGA2 (Science. 2009, 326, p1468). Investigation of the somatic mutation which causes upregulation of HMGA2 is being conducted. The dysregulation of Wnt pathway is one of the candidate mechanisms (Blood. 2009, vol.114, no.22, p786). These results are consistent with our 3-step model of PNH pathogenesis, that is, clonal expansion is caused not only by survival advantage from immunological attack but also by benign-tumor-like proliferation. Disclosures: No relevant conflicts of interest to declare.
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Aoki, Y., K. Hiromatsu, J. Usami, M. Makino, H. Igarashi, J. Ogasawara, S. Nagata, and Y. Yoshikai. "Clonal expansion but lack of subsequent clonal deletion of bacterial superantigen-reactive T cells in murine retroviral infection." Journal of Immunology 153, no. 8 (October 15, 1994): 3611–21. http://dx.doi.org/10.4049/jimmunol.153.8.3611.
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Abstract Several studies have suggested that activation-induced apoptosis of Ag-specific CD4+ T cells leads to depletion of this subset during HIV infection. The bacterial superantigen, staphylococcal enterotoxin A (SEA), is known to induce activation-induced apoptosis in the TCR V beta-bearing CD4+ T cells in the periphery after clonal expansion of these cells. The murine retroviral model of AIDS (MAIDS), which is induced by LP-BM5 murine leukemia virus, shares many common features with HIV infection in humans, except that CD4+ T cells increase progressively in susceptible strains. In this study, we challenged SEA to MAIDS mice and examined whether this retrovirus affects the fate of the SEA-reactive CD4+ T cells in vivo. At 4 wk post-infection with LP-BM5 murine leukemia virus, clonal expression and subsequent deletion of SEA-reactive CD4+V beta 3+ T cells occurred normally after SEA administration, whereas in vitro proliferative responses were severely impaired. At 8 wk postinfection, the in vivo expansion of CD4+V beta 3+ T cells was evident, but not followed by clonal deletion, as late as 14 days after SEA administration. This expanding subset in the infected mice expressed the Fas Ag in the same amount as the same subset in uninfected controls. These findings suggest that activation-induced apoptosis of superantigen-reactive CD4+ T cells is interfered with in vivo during the course of MAIDS, which is not attributable to underexpression of the Fas Ag by the CD4+ T cells.
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Heidenreich, W. F., and H. G. Paretzke. "The Two-Stage Clonal Expansion Model as an Example of a Biologically Based Model of Radiation-Induced Cancer." Radiation Research 156, no. 5 (November 2001): 678–81. http://dx.doi.org/10.1667/0033-7587(2001)156[0678:ttscem]2.0.co;2.
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Yoon, John K., Joseph R. Holloway, Daria W. Wells, Machika Kaku, David Jetton, Rebecca Brown, and John M. Coffin. "HIV proviral DNA integration can drive T cell growth ex vivo." Proceedings of the National Academy of Sciences 117, no. 52 (December 14, 2020): 32880–82. http://dx.doi.org/10.1073/pnas.2013194117.
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In vivo clonal expansion of HIV-infected T cells is an important mechanism of viral persistence. In some cases, clonal expansion is driven by HIV proviral DNA integrated into one of a handful of genes. To investigate this phenomenon in vitro, we infected primary CD4+ T cells with an HIV construct expressing GFP and, after nearly 2 mo of culture and multiple rounds of activation, analyzed the resulting integration site distribution. In each of three replicates from each of two donors, we detected large clusters of integration sites with multiple breakpoints, implying clonal selection. These clusters all mapped to a narrow region within the STAT3 gene. The presence of hybrid transcripts splicing HIV to STAT3 sequences supports a model of LTR-driven STAT3 overexpression as a driver of preferential growth. Thus, HIV integration patterns linked to selective T cell outgrowth can be reproduced in cell culture. The single report of an HIV provirus in a case of AIDS-associated B-cell lymphoma with an HIV provirus in the same part of STAT3 also has implications for HIV-induced malignancy.
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Dewanji, Anup, Jihyoun Jeon, Rafael Meza, and E. Georg Luebeck. "Number and Size Distribution of Colorectal Adenomas under the Multistage Clonal Expansion Model of Cancer." PLoS Computational Biology 7, no. 10 (October 13, 2011): e1002213. http://dx.doi.org/10.1371/journal.pcbi.1002213.
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Zeka, A., R. Gore, and D. Kriebel. "The two-stage clonal expansion model in occupational cancer epidemiology: results from three cohort studies." Occupational and Environmental Medicine 68, no. 8 (November 11, 2010): 618–24. http://dx.doi.org/10.1136/oem.2009.053983.
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Richardson, David B. "Lung cancer in chrysotile asbestos workers: analyses based on the two-stage clonal expansion model." Cancer Causes & Control 20, no. 6 (January 29, 2009): 917–23. http://dx.doi.org/10.1007/s10552-009-9297-z.
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Micevic, G., A. Daniels, K. Flem-Karlsen, R. Talty, K. Park, M. Bosenberg, and R. Flavell. "1234 Immunotherapy induces clonal expansion of cytotoxic tumor-specific TILs in a model of melanoma." Journal of Investigative Dermatology 143, no. 5 (May 2023): S212. http://dx.doi.org/10.1016/j.jid.2023.03.1248.
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Traulsen, Arne, Tom Lenaerts, Jorge M. Pacheco, and David Dingli. "On the dynamics of neutral mutations in a mathematical model for a homogeneous stem cell population." Journal of The Royal Society Interface 10, no. 79 (February 6, 2013): 20120810. http://dx.doi.org/10.1098/rsif.2012.0810.
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The theory of the clonal origin of cancer states that a tumour arises from one cell that acquires mutation(s) leading to the malignant phenotype. It is the current belief that many of these mutations give a fitness advantage to the mutant population allowing it to expand, eventually leading to disease. However, mutations that lead to such a clonal expansion need not give a fitness advantage and may in fact be neutral—or almost neutral—with respect to fitness. Such mutant clones can be eliminated or expand stochastically, leading to a malignant phenotype (disease). Mutations in haematopoietic stem cells give rise to diseases such as chronic myeloid leukaemia (CML) and paroxysmal nocturnal haemoglobinuria (PNH). Although neutral drift often leads to clonal extinction, disease is still possible, and in this case, it has important implications both for the incidence of disease and for therapy, as it may be more difficult to eliminate neutral mutations with therapy. We illustrate the consequences of such dynamics, using CML and PNH as examples. These considerations have implications for many other tumours as well.
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Chen, Huaying, Haixu Meng, Zhenlin Chen, Tong Wang, Chuanpin Chen, Yonggang Zhu, and Jing Jin. "Size-Based Sorting and In Situ Clonal Expansion of Single Cells using Microfluidics." Biosensors 12, no. 12 (November 30, 2022): 1100. http://dx.doi.org/10.3390/bios12121100.
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Separation and clonal culture and growth kinetics analysis of target cells in a mixed population is critical for pathological research, disease diagnosis, and cell therapy. However, long-term culture with time-lapse imaging of the isolated cells for clonal analysis is still challenging. This paper reports a microfluidic device with four-level filtration channels and a pneumatic microvalve for size sorting and in situ clonal culture of single cells. The valve was on top of the filtration channels and used to direct fluid flow by membrane deformation during separation and long-term culture to avoid shear-induced cell deformation. Numerical simulations were performed to evaluate the influence of device parameters affecting the pressure drop across the filtration channels. Then, a droplet model was employed to evaluate the impact of cell viscosity, cell size, and channel width on the pressure drop inducing cell deformation. Experiments showed that filtration channels with a width of 7, 10, 13, or 17 μm successfully sorted K562 cells into four different size ranges at low driving pressure. The maximum efficiency of separating K562 cells from media and whole blood was 98.6% and 89.7%, respectively. Finally, the trapped single cells were cultured in situ for 4–7 days with time-lapse imaging to obtain the lineage trees and growth curves. Then, the time to the first division, variation of cell size before and after division, and cell fusion were investigated. This proved that cells at the G1 and G2 phases were of significantly distinct sizes. The microfluidic device for size sorting and clonal expansion will be of tremendous application potential in single-cell studies.
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Tovy, Ayala, Hyun Jung Park, Jaime M. Reyes, Anna Guzman, Rachel E. Rau, Aaron Jeffries, Mill Jonnathan, et al. "Mosaic DNMT3A Germline Mutation As a Model for Mutant DNMT3A Competitive Advantage in the Blood Lineage." Blood 132, Supplement 1 (November 29, 2018): 173. http://dx.doi.org/10.1182/blood-2018-173.
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Abstract The DNA Methyltransferase 3A (DNMT3A) gene is recurrently mutated in a large spectrum of hematologic malignancies, including acute myeloid leukemia (AML). About 25% of adult AML patients carry mutations in DNMT3A and these mutations are generally associated with poor prognosis. DNMT3A mutations have been also associated with aged-related clonal hematopoiesis of indeterminate potential (CHIP). The high prevalence of DNMT3A somatic mutations in AML and CHIP implies that cells with mutated DNMT3A have a competitive advantage over wild-type (WT) cells, resulting in clonal expansion. However, the downstream molecular mechanisms that underlie this phenotype are not clear. Tatton-Brown-Raman syndrome (TBRS) is a rare genetic disorder caused by heterozygous germline mutations in DNMT3A, characterized by overgrowth and developmental delay. In one particular family, a group of 4 children out of 12 were diagnosed with TBRS and were found to be heterozygous carriers of DNMT3A-R771Q mutation (DNMT3AR771Q) inherited from their mosaic father. Thus, this individual provides a unique opportunity to study the long-term consequences of DNMT3A mutations, as he harbors both WT and mutant cells. From this mosaic individual, we generated lymphoblastoid cell lines (LCLs) from the peripheral blood (PB) and measured DNMT3AR771Q variant allele frequency (VAF) in the LCL pool as well as in PB, saliva and urine, all collected at the same time. Strikingly, DNMT3AR771Q VAF in the LCL pool and in PB was substantially higher than in saliva and urine (respectively 30%, 45%, 10%, 4%), implying that levels of DNMT3A mosaicism are tissue-specific and that cells with mutated DNMT3A tend to expand in the blood but not in epithelia (figure 1A and figure1B). One hypothesis for the prevalence of DNMT3A mutations in AML is that its loss reduces the effectiveness of DNA repair leading to increased mutational rates. In order to test this, we compared the mutational loads in individual LCL clones that were WT or DNMT3A mutant using whole genome sequencing. Surprisingly, no clear differences were observed between WT and DNMT3AR771Q mutant cells, indicating that clonal expansion is unlikely to be secondary to a general increase in mutational burden. To explore the impact of DNMT3AR771Q mutation on DNA methylation, we performed whole-genome bisulfite sequencing (WGBS) on two WT and two DNMT3AR771Q LCL clones. We identified ~31,500 differentially methylated regions (DMRs) between WT and mutant clones, with the majority of DMRs being hypomethylated. Hypomethylated DMRs were associated with gene regulatory regions, mainly promoters and enhancer regions. These data suggest that the DNMT3AR771Q mutation affects DNA methylation setting at genomic regions that can directly affect transcription. Canyons are large genomic regions of low methylation that often occur around master regulators such as homeobox-containing genes. We previously showed in mice that DNMT3A regulates DNA methylation at canyon edges, with loss of DNMT3A resulting in canyon expansion. In agreement, DNMT3AR771Q mutant clones displayed larger canyons, particularly at loci marked by H3K27Ac and H3K4me3 (figure 1C). Gene Ontology analysis of genes falling into expanded canyons showed a significant enrichment for leukemia and stem cell-related genes, including members of the HOX family. RNAseq analysis of DNMT3AR771Q mutant LCL clones confirmed the upregulation of key cancer-associated genes. These data suggest that DNMT3A mutations may promote clonal expansion through hypomethylation and overexpression of stem cell and cancer-related genes In conclusion, by comparing WT and DNMT3Amutant LCL clones generated from the same individual, we show that DNMT3A mutations lead to significant hypomethylation and overexpression of key cancer-associated genes. Further studies on specific target genes will reveal critical pathways responsible for the clonal expansion of cells with mutated DNMT3A, paving the way for the development of new therapeutic strategies for malignancies with mutated DNMT3A. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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Anderton, Stephen M., Caius G. Radu, Pauline A. Lowrey, E. Sally Ward, and David C. Wraith. "Negative Selection during the Peripheral Immune Response to Antigen." Journal of Experimental Medicine 193, no. 1 (December 27, 2000): 1–12. http://dx.doi.org/10.1084/jem.193.1.1.
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Thymic selection depends on positive and negative selective mechanisms based on the avidity of T cell interaction with antigen–major histocompatibility complex complexes. However, peripheral mechanisms for the recruitment and clonal expansion of the responding T cell repertoire remain obscure. Here we provide evidence for an avidity-based model of peripheral T cell clonal expansion in response to antigenic challenge. We have used the encephalitogenic, H-2 Au-restricted, acetylated NH2-terminal nonameric peptide (Ac1-9) epitope from myelin basic protein as our model antigen. Peptide analogues were generated that varied in antigenic strength (as assessed by in vitro assay) based on differences in their binding affinity for Au. In vivo, these analogues elicited distinct repertoires of T cells that displayed marked differences in antigen sensitivity. Immunization with the weakest (wild-type) antigen expanded the high affinity T cells required to induce encephalomyelitis. In contrast, immunization with strongly antigenic analogues led to the elimination of T cells bearing high affinity T cell receptors by apoptosis, thereby preventing disease development. Moreover, the T cell repertoire was consistently tuned to respond to the immunizing antigen with the same activation threshold. This tuning mechanism provides a peripheral control against the expansion of autoreactive T cells and has implications for immunotherapy and vaccine design.
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Curtis, Christina. "Population Genetics Approaches to Quantify Clonal Evolution." Blood 130, Suppl_1 (December 7, 2017): SCI—37—SCI—37. http://dx.doi.org/10.1182/blood.v130.suppl_1.sci-37.sci-37.
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Abstract Cancer results from the acquisition of somatic alterations in an evolutionary process that typically occurs over many years, much of which is occult. Understanding the evolutionary dynamics that are operative at different stages of progression in individual tumors might inform the earlier detection, diagnosis, and treatment of cancer. For decades, tumor progression has been described as a gradual stepwise process, and it is through this lens that the underlying mechanisms have been interpreted and therapeutic strategies have been developed. Although these processes cannot be directly observed, the resultant spatiotemporal patterns of genetic variation amongst tumor cells encode their evolutionary histories. Cancer genome sequencing has thus yielded unprecedented insights into intra-tumor heterogeneity (ITH) and these data enable the inference of tumor dynamics using population genetics techniques. The application of such approaches suggests that tumor evolution is not necessarily gradual, but rather can be punctuated, resulting in revision of the de facto sequential clonal expansion model. For example, we previously described a Big Bang model of human colorectal tumor growth, wherein after transformation the neoplasm grows predominantly as a single terminal expansion in the absence of stringent selection, compatible with effectively neutral evolution1. In the Big Bang model, the timing of a mutation is the fundamental determinant of its frequency in the final tumor such that all major clones persist during growth and most detectable intra-tumor heterogeneity (ITH) occurs early. By analyzing multi-region and single gland genomic profiles in colorectal adenomas and carcinomas within a spatial agent-based tumor growth model and Bayesian statistical inference framework, we demonstrated the early origin of ITH and verified several other predictions of the Big Bang model. This new model provides a quantitative framework for understanding tumor progression with several clinical implications. In particular, rare but potentially aggressive subclones may be undetectable, providing a rich substrate for the emergence of resistance under treatment selective pressure. These data also suggest that some tumors may be born to be bad, wherein malignant potential is specified early. While not all tumors exhibit Big Bang dynamics, effectively neutral evolution has since been reported in other tumors and hence may be relatively common. These findings emphasize the need for methods to infer the role of selection in established human tumors and the systematic evaluation of distinct modes of evolution across tumor types and disease stages. To address this need, we developed an extensible population genetics framework to simulate spatial tumor growth and evaluate evidence for different evolutionary modes based on patterns of genetic variation derived from multi-region sequencing (MRS) data2. We demonstrate that while it is feasible to distinguish strong positive selection from neutral tumor evolution, weak selection and neutral evolution were indistinguishable in current data. Building on these findings, we developed a classifier that exploits novel measures of ITH and applied this to MRS data from diverse tumor types, revealing different evolutionary modes amongst treatment naïve tumors. To better understand evolutionary tempos during disease progression, we further characterized longitudinally sampled specimens. These findings have implications for forecasting tumor evolution and designing more effective treatment strategies. 1. Sottoriva A, Kang H, Ma Z, et al. A Big Bang model of human colorectal tumor growth. Nature Genetics. 2015;47:209-16. 2. Sun R, Hu Z, Sottoriva A, et al. Between-region genetic divergence reflects the mode and tempo of tumor evolution. Nature Genetics. 2017;49:1015-24. Disclosures No relevant conflicts of interest to declare.
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Shindyapina, Anastasia, Jose Castro, Alessandro Barbieri, João Paulo, Olga Strelkova, John Sedivy, John Manis, and Vadim Gladyshev. "Aging Predisposes B cells to Malignancy by Activating c-Myc and Perturbing the Genome and Epigenome." Innovation in Aging 5, Supplement_1 (December 1, 2021): 560–61. http://dx.doi.org/10.1093/geroni/igab046.2152.
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Abstract Age is the single major risk factor for human cancer, but naturally occurring cancers are rarely studied in aging models. Like humans, mice spontaneously develop cancer with age, and standard laboratory strains are predisposed for B-cell lymphoma. Here, we uncover how B-cell lymphoma develops as a consequence of the aging immune system. We found that aged B cells acquire somatic mutations in tumor suppressors and oncogenes (e.g. Trp53, Pim1, and Myh11) and undergo monoclonal expansions, with some clones representing 86% of splenic B cells. Clonal B cells had hypermethylated promoters and globally silenced expression, suggesting a role of DNA methylation in clonal selection of premalignant B cells. B-cell size, spleen weight, and a novel population of B cells, which we named Myc+ cells, emerged as convenient markers of malignancy. High-throughput analyses of clonal B cells and the use of genetic mouse models revealed that c-Myc drives B-cell size increase and clonal expansion with age. Phoshoproteome and co-culture experiments revealed that c-Myc is activated by signals from the aging microenvironment. Moreover, single-cell RNA-seq suggested that clonal B cells originate from age-associated B cells, further underlying the importance of aging environment in cancer transformation. Longitudinal analyses demonstrated a negative impact of premalignant B cells on mouse lifespan and linked it to age-related myeloid bias. Together, our study revealed cell-autonomous changes that cooperate with the aging microenvironment to give rise to preneoplastic B cells. This stidy established a novel model to study how aging predisposes cells to cancer transformation.
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Bond, J. A., M. F. Haughton, J. M. Rowson, P. J. Smith, V. Gire, D. Wynford-Thomas, and F. S. Wyllie. "Control of Replicative Life Span in Human Cells: Barriers to Clonal Expansion Intermediate Between M1 Senescence and M2 Crisis." Molecular and Cellular Biology 19, no. 4 (April 1, 1999): 3103–14. http://dx.doi.org/10.1128/mcb.19.4.3103.
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ABSTRACT The accumulation of genetic abnormalities in a developing tumor is driven, at least in part, by the need to overcome inherent restraints on the replicative life span of human cells, two of which—senescence (M1) and crisis (M2)—have been well characterized. Here we describe additional barriers to clonal expansion (Mint) intermediate between M1 and M2, revealed by abrogation of tumor-suppressor gene (TSG) pathways by individual human papillomavirus type 16 (HPV16) proteins. In human fibroblasts, abrogation of p53 function by HPVE6 allowed escape from M1, followed up to 20 population doublings (PD) later by a second viable proliferation arrest state, MintE6, closely resembling M1. This occurred despite abrogation of p21WAF1 induction but was associated with and potentially mediated by a further ∼3-fold increase in p16INK4a expression compared to its level at M1. Expression of HPVE7, which targets pRb (and p21WAF1), also permitted clonal expansion, but this was limited predominantly by increasing cell death, resulting in a MintE7 phenotype similar to M2 but occurring after fewer PD. This was associated with, and at least partly due to, an increase in nuclear p53 content and activity, not seen in younger cells expressing E7. In a different cell type, thyroid epithelium, E7 also allowed clonal expansion terminating in a similar state to MintE7 in fibroblasts. In contrast, however, there was no evidence for a p53-regulated pathway; E6 was without effect, and the increases in p21WAF1 expression at M1 and MintE7 were p53 independent. These data provide a model for clonal evolution by successive TSG inactivation and suggest that cell type diversity in life span regulation may determine the pattern of gene mutation in the corresponding tumors.
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Schwartz, Logan Sari, Kristina Mujica, Rebecca Bell, Matthew Loberg, and Jennifer Trowbridge. "Defining Germline Predisposition to Dnmt3a-Mutant Clonal Hematopoiesis (CH)." Blood 134, Supplement_1 (November 13, 2019): 1198. http://dx.doi.org/10.1182/blood-2019-124530.
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Clonal hematopoiesis (CH) is the result of somatic mutations that confer a selective advantage to hematopoietic stem cells (HSCs), driving them to outcompete other HSCs and dominate mature hematopoietic cell production. While it is estimated that by middle age nearly all healthy individuals will have low but detectable variant allele frequency (VAF) indicating CH, only a small fraction will progress to hematologic malignancy. No methods currently exist to predict who will or will not progress to hematologic malignancy and preventative therapeutic strategies are lacking, largely due to poor understanding of the risk factors that determine whether individuals are susceptible to, or protected from, CH. Several recent population sequencing studies have found that inherited genetic variants increase the likelihood of developing CH and that there are population differences in clonal advantages gained by specific mutations in particular genetic and environmental contexts. Limitations of these human studies are the insufficient ability to control for and distinguish genetic from environmental contributions to CH, and to directly interrogate mechanisms causing genetic predisposition to CH and progression to malignancy. To overcome these barriers, here we have utilized the eight inbred and wild-derived founder strains of the Collaborative Cross (CC) and Diversity Outbred (DO) mouse populations (Saul MC et al., Trends in Genetics 2019). These feature well-characterized, segregated genetic variation that accurately reflects the genetic structure of human populations in a controlled environment. We crossed our recently described mouse model of CH (Dnmt3aR878H/+Mx1-Cre on a C57BL/6 background) (Loberg MA et al., Leukemia 2019) with each of the eight founder strains. We observed that genetic background conferred sensitivity or resistance to Dnmt3aR878H/+ HSC expansion based on phenotypic cell surface markers (Lin- c-Kit+ Sca-1+ CD150+ CD48-), relative to Dnmt3a+/+Mx1-Cre strain littermate controls. Genetic contribution from the C57BL/6 and CAST strains permitted robust Dnmt3aR878H/+ HSC expansion compared to Dnmt3a+/+ controls over a period of 6 months. In this same time frame, genetic contribution from 129, NOD, NZO, A/J, PWK, WSB strains demonstrated a spectrum of resistance to Dnmt3aR878H/+ HSC expansion. In vitro studies using colony-forming assays of bone marrow cells isolated from selected strains show a positive correlation between serial replating efficiency and phenotypic HSC frequency, supporting that phenotypic cell surface markers reliably define cells with functional potential across these strains. Ongoing studies to interrogate in vivo functional potential by competitive transplantation of HSCs isolated from each of these strains will be discussed. Together, our work demonstrates that HSC expansion as a consequence of Dnmt3a mutation is influenced by heritable genetic background in a controlled environmental setting. Understanding the mechanisms, at the systemic, intracellular and/or molecular levels, by which genetic background modifies HSC expansion caused by Dnmt3a or other CH mutations will be critical to develop improved prognostic tools and therapeutics to diagnose and treat high-risk versus low-risk CH. For this, utilization of genetically diverse mouse populations provides a critically needed platform to determine causative mechanisms driving clonal expansion of HSCs. Disclosures Trowbridge: Fate Therapeutics: Patents & Royalties: patent license.
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Vela, Pablo Sánchez, Anthony Martinez Benitez, Aishwarya Krishnan, Maria Kleppe, Sheng F. Cai, and Ross L. Levine. "Abstract P073: Targeting JAK1 signaling for molecular prevention in clonal hematopoiesis." Cancer Prevention Research 16, no. 1_Supplement (January 1, 2023): P073. http://dx.doi.org/10.1158/1940-6215.precprev22-p073.
Full textAbstract:
Abstract Myeloid malignancies are characterized by the stepwise acquisition of different somatic mutations in hematopoietic stem and progenitor cells (HSPCs) that promote subsequent leukemic transformation. When these mutations, including in the epigenetic regulator TET2, are found in the blood cells of individuals without any signs of hematologic malignancy, this condition is termed clonal hematopoiesis (CH). The incidence of CH increases with age and has been recognized as a risk factor for the development of secondary heme malignancies and cardiovascular disease. Nonetheless, currently no therapies exist to alter the natural course of CH. Accumulating evidence indicates that inflammatory signals can enhance myeloproliferation of CH HSPCs suggesting a key role of inflammatory stressors in the promotion of the clonal advantage of Tet2-mutant stem cells. However, whether hijacking this inflammatory signaling will prevent clonal expansion and leukemogenesis is currently unknown. The members of the Janus family of nonreceptor tyrosine kinases (JAK) transmit a diversity of ligand-mediated signals and act as a signaling-hub for inflammation. Our central hypothesis is that CH and progression to acute myeloid leukemia (AML) occurs in the setting of inflammatory stress and that mutant clonal expansion is mediated by JAK/STAT inflammatory signaling, primarily through JAK1, a non-essential gene in adult hematopoiesis. We previously showed that Jak1 is critical for stress hematopoiesis in HSPCs. To assess whether Tet2-mediated clonal expansion requires Jak1, signaling, we established a conditional Scl driven Cre-inducible deletion model of Tet2-/- and Jak1-/-. We have adapted a bone marrow derived endothelial cell organoid system that allows to maintain and expand HSPCs during longer periods of time. In this setting Tet2-/- HSPCs show increased sensitivity to IL3, a Jak1-dependent cytokine that mediates exit of quiescence in HSPCs. The loss of competitive advantage of Tet2-/- Jak1-/- cells persists even in the context of co-culture with Jak1-/- Tet2-wildtype cells, and to a lesser extent in the presence of the Jak1 inhibitor Itacitinib, suggesting a denser requirement for Jak1 in CH mutant clones compared to wild-type HSPCs. Ex-vivo colony forming assays and in vivo competitive transplants demonstrate that the self-renewal abilities of Tet2-mutant HSPCs require Jak1 signaling. Furthermore, the extramedullary hematopoiesis observed in a primary model of Tet2-/- pre-leukemic myeloproliferation, was also dependent on Jak1. Moreover, studies in Tet2-/-/Flt3ITD and Mll-AF9 AML models showed that pharmacologic Jak1 inhibition abrogated ex vivo colony formation in both AMLs. This study is significant because targeting JAK/STAT mediated inflammatory signaling in CH-mutant HSPCs has the ability to translate into a precision interception strategy aimed at preventing clonal expansion and leukemic transformation. Citation Format: Pablo Sánchez Vela, Anthony Martinez Benitez, Aishwarya Krishnan, Maria Kleppe, Sheng F. Cai, Ross L. Levine. Targeting JAK1 signaling for molecular prevention in clonal hematopoiesis. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P073.
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Benoun, Joseph, and Zachary Fogassy. "Examining pathogen specific T cell responses during antibiotic intervention (MPF2P.743)." Journal of Immunology 194, no. 1_Supplement (May 1, 2015): 63.2. http://dx.doi.org/10.4049/jimmunol.194.supp.63.2.
Full textAbstract:
Abstract Salmonella enterica serovar Typhi can cause recurrent and relapsing infection in antibiotic-treated individuals, suggesting that bacterial clearance may hinder the development of adaptive immunity. We have developed an antibiotic-treatment model in mice to examine this issue using antigen-specific reagents. Infection with Salmonella Typhimurium strain BRD509-2W1S caused expansion of 2W1S-specific CD4 T cells that were easily detected in peripheral blood and lymphoid tissues. In contrast, antibiotic-treated mice displayed lower 2W1S-specific CD4 clonal expansion in blood and lymphoid tissues that persisted for months after infection. This reduced clonal frequency correlated with diminished protective immunity to re-challenge infection. We are currently examining the phenotype and functional differences in CD4 T cell memory populations to determine if central, effector, and tissue-resident memory cells are equally impacted by antibiotic-intervention. Greater understanding of how antibiotics affect CD4 memory development may allow for therapeutics to boost protective immunity to secondary or relapsing Salmonella infections.
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Mueller, D. L., L. Chiodetti, P. A. Bacon, and R. H. Schwartz. "Clonal anergy blocks the response to IL-4, as well as the production of IL-2, in dual-producing T helper cell clones." Journal of Immunology 147, no. 12 (December 15, 1991): 4118–25. http://dx.doi.org/10.4049/jimmunol.147.12.4118.
Full textAbstract:
Abstract In this report we extend the in vitro clonal anergy model to examine the regulation of proliferation in T cells that secrete both IL-2 and IL-4. Newly cloned Ag-specific murine T cells are shown to depend on both IL-2 and IL-4 synthesis for maximal proliferation. Whereas IL-2 responsiveness is constitutive in these cells, IL-4 responsiveness develops only after Ag and APC stimulation. Remarkably, proliferation of these cells to Ag is sensitive to inhibition by clonal anergy, even though IL-4 synthesis remains inducible. Anergy in these cells is associated with an inability to respond to IL-4, in addition to the development of an IL-2 production defect. The results suggest that anergy induction may be capable of preventing the clonal expansion of autoreactive T cells producing both IL-2 and IL-4 in vivo.
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Bhaumik, Siddhartha, Sunil Thomas, Albanus Moguche, Michael Wijaranakula, and Murali-Krishna Kaja. "The anti-viral and signal-3 effects of Type-I interferons exert opposing effects on clonal expansion of CD8 T cells during West Nile Virus infection (154.13)." Journal of Immunology 186, no. 1_Supplement (April 1, 2011): 154.13. http://dx.doi.org/10.4049/jimmunol.186.supp.154.13.
Full textAbstract:
Abstract West Nile Virus (WNV) is a neurotropic positive stranded RNA virus, which affects central nervous system in a fraction of infected humans in several continents, including North America. In concert with innate responses both antibody and T cell mediated immunity play an important role in conferring protection against WNV. While the importance of type-I IFN-I (IFN-I) signaling on WNV-specific innate immunity is well documented, it remains unclear whether and what effects IFN-I have on WNV-specific adaptive response. Using a murine foot pad infection model of WNV infection here we asked two questions: (1) what happens to virus-specific CD8 T cell responses if the infected host is devoid of IFN-I signaling, (2) what happens to virus-specific CD8 response if only CD8 T cells lack IFN-I signaling. We found that lack of IFN-I signaling in the host results in exaggerated CD8 clonal expansion whereas lack of IFN-I signaling exclusively in CD8 T cells results in diminished clonal expansion. Our results together, suggest INF-I mediated innate viral control and the ability to provide signal-3 exerts opposing effects on CD8 T cell expansion during West Nile Virus infection.
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Loberg, Matthew, Rebecca Bell, Tim Stearns, Leslie Goodwin, Kira Young, David Bergstrom, Rebekka Schneider, and Jennifer J. Trowbridge. "Mutation in DNA Methyltransferase DNMT3A Confers Enhanced Self-Renewal Capacity Onto Multipotent Progenitor Cells and Predisposes to Acute Myeloid Leukemia (AML)." Blood 132, Supplement 1 (November 29, 2018): 2569. http://dx.doi.org/10.1182/blood-2018-99-111560.
Full textAbstract:
Abstract The clinical significance of clonal hematopoiesis (CHIP or ARCH) remains a barrier to predicting the risk of hematologic malignancy. DNMT3A is a de novo DNA methyltransferase frequently mutated in clonal hematopoiesis, myelodysplastic syndrome and acute myeloid leukemia. Loss of or mutation in DNMT3A has been demonstrated to enhance self-renewal of hematopoietic stem cells (HSCs), suggesting that this is the predominant cell population driving clonal hematopoiesis. How DNMT3A-mutant cells become at risk for transformation is unclear, in part due to our limited understanding of how DNMT3A mutation confers a selective advantage and the cooperating mechanisms required for progression to MDS or AML. To address this gap in knowledge, we generated a cre-inducible Dnmt3a-LSL-R878H mouse model (representing the DNMT3A-R882H mutation commonly found in human AML), in which wild-type Dnmt3a expression is preserved prior to recombination. Heterozygous Dnmt3aR878H mice exhibit an expansion in both HSCs and multipotent progenitor (MPP) cell subsets with distinct kinetics. Transcriptional profiling of sorted HSC and MPP populations by RNA-seq revealed distinct transcriptional signatures indicating that different mechanisms underlie expansion of Dnmt3aR878H/+ HSCs and MPPs. Dnmt3a-mutant HSCs exhibit downregulation of genes important for differentiation, while Dnmt3a-mutant MPPs exhibit upregulation of genes associated with stem cell self-renewal, including Jam2 and Ryk. Functionally, we observe that Dnmt3a-mutant MPPs have enhanced serial replating capacity in in vitro colony assays. These data suggest that mutation in DNMT3A may cause clonal hematopoietic expansion through distinct mechanisms dependent on the cell-of-origin which incurs this mutation. To determine whether clonal hematopoiesis driven by Dnmt3aR878H/+ was sufficient to predispose to a hematologic malignancy, we generated an independent, Flp-inducible Npm1-FSF-cA mouse model (representing the NPM1cA mutation commonly found in human AML), in which wild-type Npm1 expression is preserved prior to recombination. Inducing Npm1cA mutation in hematopoietic stem and progenitor cells carrying Dnmt3aR878H caused development of a fully penetrant myeloproliferative disorder upon transplant into recipient mice. Transplantation of these cells into secondary recipient mice led to a fully penetrant AML with accelerated disease kinetics compared to primary transplant recipients. These data suggest that the combination of DNMT3A mutation followed by NPM1 mutation is sufficient to cause AML. In summary, this study reveals a novel cell context-specificity of how DNMT3A mutation confers a selective advantage and demonstrates that NPM1 mutation can cooperate with DNMT3A mutation to cause AML. This work has implications for predicting individuals at risk of progression from clonal hematopoiesis to AML. Disclosures No relevant conflicts of interest to declare.