Lamis Naddaf’s research while affiliated with The Jackson Laboratory and other places

The incidence of clonal hematopoiesis of indeterminate potential (CHIP) increases exponentially with aging. Still, rare clones harboring the prototypical clonal hematopoiesis (CH) mutations are nearly ubiquitous in middle aged individuals, suggesting that cell intrinsic or micro-environmental changes observed with aging can promote the expansion of malignant clones to pathologically relevant levels, increasing the risk of hematological malignancies and a multitude of sequelae associated with CHIP. To address if aging can alter the fitness dynamics regulating the rate of expansion of malignant clones, we utilized a CRISPR based approach in hematopoietic stem and progenitor cells (HSPC) from mice of different ages to introduce mutations in the Tet2 and Dnmt3a genes or in the Rosa26 safe harbor locus (as competitors), and analyzed their rate of expansion after transplantation in aged matched mice. Our data show that Dnmt3a KO cells expand with similar rates in young and old recipient mice after primary and secondary transplant. In contrast, Tet2 KO cells expand at a significantly faster rate in old HSPC in old mice (20 months old) in comparison to young HSPC in young mice (2 months old) and in all hematopoietic populations in the peripheral blood and the bone marrow. Importantly, by heterochronic transplantation of young or old donor HSPC in old or young recipient mice respectively, we observe that the rate of expansion of the Tet2 mutant cells is solely determined by the age of the donor, suggesting that cell intrinsic age-associated changes in the HSPC population facilitate the expansion of the Tet2 KO clones. By using donor mice of different ages, we observed that the rate of expansion of the Tet2 KO cells is significantly accelerated by 7 months of age in both male and female mice. Aging is associated with shifts in the relative frequencies of HSPC sub-populations - therefore we sorted myeloid biased HSC, lymphoid biased HSC, short-term (ST) HSC and multipotent progenitor cells (MPP) from old mice, and after Tet2 CRISPR, transplanted the cells into young recipient mice. Both myeloid and lymphoid biased HSC show a similar accelerated rate of Tet2 KO cell expansion, suggesting that the increased myeloid bias with age is not correlated with the Tet2 KO adaptation. In ST-HSC and MPP, we did not observe a selection for the Tet2 clones in the short term. As expected, we noticed a dwindling engraftment in mice transplanted with control ST-HSC and MPP with time; however, Tet2 KO cells exhibited much more robust and prolonged maintenance in these multi-potent progenitor populations, suggesting that the loss of Tet2 can extend the limited self-renewal of progenitor cells. Next, we performed competitive transplantations with donor cells with differing genotype (WT or Tet2 KO) or age (2 months or 20 months) to directly compare their relative fitness. Our results show that young and old Tet2 KO HSPC have similar fitness levels, and young Tet2 KO HSPC having a small advantage over young control cells. In contrast, young WT HSPC have a much larger fitness advantage than old WT HSPC. Thus, the enhanced Tet2 KO expansion with age is due to an intrinsic defect of the WT population that cannot effectively compete with Tet2 mutant clones. To elucidate the molecular mechanisms for the fitness advantage of Tet2 KO cells with aging, we performed scRNAseq on WT or Tet2 KO HSPC from 2, 12 and 20 month old mice isolated 12 weeks after transplantation into age matched recipient mice. By performing GSEA analyses, we found that aging increased signatures related to mTORC1 activity, ribosomal biogenesis and mitochondrial oxidative phosphorylation, as well as pathways related to RUNX1 and p53 activity, all of which were reverted in Tet2 KO cells to young-like levels. In conclusion, our study supports the hypothesis that TET2 mutations observed in CH are selectively advantageous in aging due to cell intrinsic changes occurring in HSC. While the specific age-associated alteration(s) in HSC leading to the adaptation of TET2 mutant clones remain to be defined, we find that several transcriptional changes observable with aging are reverted by the loss of Tet2, likely endowing mutant cells with a larger fitness differential than that observable in a young, highly fit HSC population. Interventions aimed at maintaining HSC clones with higher fitness in aging can be a powerful preventative factor against the emergence of CHIP and its negative consequences.

Aging is a process of systemic deterioration and the most significant risk factor for cancers. Clonal hematopoiesis (CH) commonly occurs with aging and links to higher mortality, leukemia risk, and cardiovascular diseases. Age-related CH involves the abnormal clonal expansion of hematopoietic stem cells (HSCs) bearing somatic mutations in genes frequently mutated in leukemia, including genes encoding epigenetic regulators, such as the DNA demethylase TET2. While such mutations are known to alter the HSC epigenome, the mechanisms through which these mutations drive HSC self-renewal, myeloid transformation, and inflammatory response remain elusive. In this study, we hypothesize that aging and CH mutations cooperatively reshape the HSC transcriptomic landscape and enhancing HSC competitive advantage that facilitates clonal expansion. Recently, aging has been closely associated with Tet2 mutation impact. Using single-cell multi-omic analyses and flow-cytometric phenotyping, we demonstrated, for the first time, that HSC aging processes at transcriptomic, epigenomic, and cellular levels are mitigated by Tet2 deficiency in mice, in an age-dependent manner. at ages greater than or equal to 16 months, but not at a young age, based on gene expression and chromatin accessibility at a single-cell resolution. This age mitigation was further validated by differential analysis of gene expression, open chromatin accessibility and DNA methylation that are elevated with aging but lowered with Tet2 deficiency in HSCs from old mice. Moreover, we found that Tet2 deficiency , a cellular hallmark of HSC aging, in an age-dependent manner. We observed increased gene expression with aging, such as for Eya4 and Lars2, which are downregulated by Tet2∆/∆ in HSCs from older mice, suggesting that Tet2 deficiency mitigates the process of aging. Also, the increased expression of Cdk6, Msi2, and Sox4 in Tet2∆/∆ HSCs, normally reduced in old HSC, promote the HSC self-renewal as shown in earlier. These gene expression changes were confirmed by single-nuclear open chromatin accessibility analysis, suggesting that Tet2 deficiency mitigates epigenome reprograming during HSC aging, as shown by increased differential single-nuclear chromatin accessibility of their motifs via ChromVar analysis. These TFs' target genes play critical roles in HSC commitment to progenitors, hemostasis, and proliferation. The methylation status of these TF binding motifs is linked to the regulation for the targets' gene expression. In conclusion, our findings reveal that Tet2 deficiency significantly contributes to the mitigation of HSC aging and hijacks the HSC expansion strategy during HSC aging via epigenetic reprogramming, which contributes to age-related clonal hematopoiesis. By elucidating the transcriptomic and epigenomic alterations in Tet2-deficient HSCs, our study provides novel insights into how age and somatic mutations interact to promote the pathogenesis of age-related hematological diseases. These discoveries not only enhance our understanding of HSC aging mechanisms but also offer potential biomarkers for early detection and targets for therapeutic intervention in age-associated clonal disorders.

Aging impacts the bone marrow microenvironment by inducing inflammation. We hypothesize that aging increases heritable epigenetic heterogeneity, leading to transcriptome heterogeneity within hematopoietic stem and progenitor cells (HSPC). This resulting heterogenous state, combined with the aged microenvironment-driven selection, shifts the functional capacity of the hematopoietic system, contributing to the functional decline of the hematopoietic system as well as the evolution of diseases such as myelodysplastic syndrome and leukemias. Our research aims to investigate clonal heterogeneity in both young and old mice and highlight the phenotypic patterns selected in each case. We employed CellTag indexing and single-cell RNA sequencing to track HSPC, identify the selected clones in young and old mice BM, and characterize their molecular signatures. We generated 10x Genomics single-cell RNA-seq for tagged HSPC after 15-day culturing (in vitro) and two months post-transplantation into mice (in vivo). Tracing the expansion of HSPC clones in young and old BM revealed critical changes in HSPC heterogeneity and selection in older mice. We observed an increase in transcriptional heterogeneity of old HSPC as compared to young samples (P<0.01, Wilcox test). Genes with higher gene expression variability in older mice coincide with pathways pivotal for HSPC fitness, e.g., ribosome synthesis, inflammation response mechanisms (JAK-STAT), hematopoietic cell lineage, AML, and cancer pathways (FDR<0.1, GSEA). At the clonal composition level, young HSPCs exhibited more oligoclonal reconstitution than old HSPC, indicated by lower Shannon entropy in young HSPCs compared to old (P<0.05 Wilcox test). This result suggests stricter clonal quality control in the young microenvironment, potentially leading to increased purifying selection that removes clones with significant transcriptional profile alterations and reduced fitness. Moreover, comparing the transcriptional profiles of clones showing robust expansion after transplantation (selected or “winner” clones) with negatively selected clones revealed several common pathways shared between young and old selected clones after expansion (FDR<0.1, GSEA), including cell cycle activity, stress response, WNT signaling, and metabolism - identifying candidate mediators of HSPC fitness in vivo. However, major differences between old and young selected clones were evident before expansion. In young, selected clones, signatures related to inflammatory response were observed.Strikingly, we observed a significant positive expression correlation (P<0.0001, Wilcox test) among the selected clones only in young mice, not in old mice. The high transcriptomic similarity among selected clones is consistent with strict clonal quality control in the young BM microenvironment. Notably, we traced the selected clones back among the tagged HSPCs in vitro (scRNA-seq) and observed higher transcriptomic similarity among selected clones from young vs. old donor mice even before transplantation (P<0.0001, Wilcox test). Collectively, these results underscore the selective nature of the young BM, contrasting with the decline of HSPC quality control in old BM, contributing to functional hematopoietic decline and fueling pre-malignant evolution. These studies reveal striking differences in clonal dynamics and quality control among HSPC in young and old mice. Clonal tracing of hematopoietic clones provides insight into how clonal competition and microenvironmental selection can contribute to hematopoietic decline and malignant evolution. Better understanding of the pathways that contribute to HSPC fitness and functional decline with aging could lead to approaches to track and perhaps even counter these aging-associated changes, which is expected to contribute to improved hematopoietic function and reduced risk of malignancy in the elderly.

Cancers are diseases of aging. Many cellular processes characteristic of cancer, e.g., epigenetic dysregulation, are similarly observed during aging. Acute myeloid leukemia (AML) is emblematic of this interplay. As a blood cancer stemming from hematopoietic stem and progenitors (HSPC), its median age of diagnosis is 65. AML is linked to age-related clonal hematopoiesis (CH), a condition resulting from the expansion of HSPC, marked by mutations in leukemia-associated genes. We aim to clarify the intrinsic role of aging in HSPC as it causes CH and leukemic transformation. Hypothesis: aging amplifies the heritable phenotypic variations in hematopoietic stem cells (HSC), enhancing epigenetic heterogeneity. This acts as a substrate for natural selection, driving leukemic transformation. Results: We found a significant increase in epigenetic and transcriptomic heterogeneity in long-term HSC (LT-HSC) from old mice compared to their younger counterparts via single-cell RNA-seq (scRNA-seq)s. This process occurs in an aged bone marrow with higher inflammation and altered HSPC support. Of relevance, we previously show that the rise of epigenetic and transcriptomic heterogeneity in HSC with Tet2 loss and Flt3 ITD precedes leukemic transformation. We further employed CellTag Indexing and scRNA-seq to track the fate of specific HSC subclones. We tracked HSPC cell culture (in vitro) after two-weeks and two-months following transplantation into recipient mice of matching ages (in vivo). Cells of the same clone manifest higher transcriptomic similarities than those from different clones, validating CellTag in mapping clonal evolution. We computed the progeny output activity (Ai) by the ratio of clone i abundance in the committed progenitors to its frequency in the HSC compartment. We found that young mice exhibit significantly higher variability and a higher mean of progeny output activity Ai, indicating the efficiency in self-renewal and cell differentiation. While both young and old mice have low-output (higher stemness) clones, only young mice had significant differential expression of key genes in low-output clones compared to high-output clones, e.g., Dpp4, Eps8, and Col5a1. Depite lower progeny output Ai variation, LT-HSCs from older mice exhibited greater clonal transcriptome diversification both in vitro and in vivo , evidenced by intra-clonal transcriptomic correlation. This pattern also existed in ST-HSC and MPP from older mice. Genes with higher expression variability in older mice highlighted pathways pivotal for HSPC fitness, e.g., ribosome synthesis (RS), inflammation response mechanisms (JAK-STAT), hematopoietic cell lineage, AML and cancer pathways (GSEA). The RS pathway were remarkably enriched in the megakaryocytic-erythroid progenitors, directly before differentiation where substantial protein translation is required. Conclusion: our results suggest that the synergy of aging and somatic mutations in epigenetic regulators may enhance cell-to-cell variations, offering additional heritable phenotypic variations. These, in turn, may facilitate clonal hematopoiesis and leukemic transformation. Future work will delineate specific epigenetic and transcriptomic configurations for mutant HSPC clonal expansion. This study provides a cohesive framework, linking the principles governing aging to cancer. Citation Format: Lamis Naddaf, Marco De Dominici, Xiaowen Chen, Travis Roeder, Hideyuki Oguro, Shilpita Karmakar, Eric Pietras, James DeGregori, Sheng Li. Accelerated intra-clonal transcriptomic diversification of hematopoietic stem cells during aging fuels leukemia evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB227.

Cancer are diseases of aging. Many cellular processes characteristic of cancer, e.g., epigenetic dysregulation, are similarly observed during aging. Acute myeloid leukemia (AML) is emblematic of this interplay. As a blood cancer stemming from hematopoietic stem and progenitors (HSPC), the median age of AML diagnosis is 65. This is linked to age-related clonal hematopoiesis (CH), a condition resulting from the expansion of hematopoietic stem and progenitor cells, typically marked by mutations in leukemia-associated genes. Our investigation thus seeks to clarify the intrinsic role of aging in HSPCs as they relate to CH and leukemic transformation. Hypothesis: Aging amplifies the inheritable phenotypic variations in hematopoietic stem cells, enhancing epigenetic heterogeneity. This acts as a substrate for natural selection, driving leukemic transformation. HSPC Epigenomic and transcriptomic heterogeneity increase in old age: we profiled the single-cell transcriptome (scRNA-seq) and single-nuclear ATAC-seq (snATAC-seq) data from Lineage-c-Kit+ HSPCs isolated from mice at two age groups. Applying Shannon entropy, our analysis revealed a significant increase in epigenetic and transcriptomic heterogeneity in middle-aged LT-HSC compared to their younger counterparts. This process occurs in an aged bone marrow (BM) microenvironment characterized by inflammation and altered HSPC support. Interestingly, we previously show that the rise of epigenetic and transcriptomic heterogeneity in HSC with Tet2 loss (Tet2 MT) and Flt3 ITD precedes leukemic transformation. Diversification of LT-HSC Transcriptome: We employed CellTag Indexing to track the fate of specific HSC subclones. With a barcode detection rate of 80%, our findings indicate that a significant majority (97%) of clones emerged from an HSC. Furthermore, we observed that cells of the same clone manifest higher transcriptomic similarities than those from different clones, underscoring the technique's capability in mapping HSPC clonal evolution. Then we applied scRNA-seq to tagged HSPCs after 16 days of ex vivo expansion. We found that LT-HSCs from older mice exhibited greater clonal diversification, evidenced by intra-clonal transcriptomic correlation. ST-HSC and MPP from older mice also exhibt this pattern. Functional Relevance: Our GSEA analysis of genes with increased expression variability in older mice highlighted several pathways pivotal for HSPC fitness, encompassing ribosome synthesis, inflammation response mechanisms (e.g., JAK-STAT), hematopoietic cell lineage, and AML/cancer pathways. Conclusion: our results suggest that the combined effects of aging and somatic mutations in epigenetic regulators may enhance cell-to-cell variations, offering additional heritable phenotypic variations. These, in turn, may facilitate clonal hematopoiesis and leukemic transformation. Future work will delineate specific epigenetic and transcriptomic configurations that enable mutant HSPC clonal expansion. This study provides a cohesive framework, linking the principles governing aging to cancer. Citation Format: Lamis Naddaf, Marco De Dominici, Xiaowen Chen, Parveen Kumar, James Chavez, Travis Roeder, Shilpita Karmakar, Eric Pietras, Hideyuki Oguro, James DeGregori, Sheng Li. Accelerated intra-clonal diversification of hematopoietic stem cells during aging fuels leukemia evolution [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B031.