Erratum: Hematologic DNMT3A reduction and high-fat diet synergize to promote weight gain and tissue inflammation.

[This corrects the article DOI: 10.1016/j.isci.2024.109122.].

Clonal evolution of hematopoietic stem cells after cancer chemotherapy.

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

SOD1 is a synthetic-lethal target in PPM1D-mutant leukemia cells.

The DNA damage response is critical for maintaining genome integrity and is commonly disrupted in the development of cancer. PPM1D (protein phosphatase Mg2+/Mn2+-dependent 1D) is a master negative regulator of the response; gain-of-function mutations and amplifications of PPM1D are found across several human cancers making it a relevant pharmacological target. Here, we used CRISPR/Cas9 screening to identify synthetic-lethal dependencies of PPM1D, uncovering superoxide dismutase-1 (SOD1) as a potential target for PPM1D-mutant cells. We revealed a dysregulated redox landscape characterized by elevated levels of reactive oxygen species and a compromised response to oxidative stress in PPM1D-mutant cells. Altogether, our results demonstrate a role for SOD1 in the survival of PPM1D-mutant leukemia cells and highlight a new potential therapeutic strategy against PPM1D-mutant cancers.

Dose-dependent effects of Dnmt3a in an inducible murine model of KrasG12D-driven leukemia.

DNMT3A mutations are frequently found in clonal hematopoiesis and a variety of hematologic malignancies, including acute myeloid leukemia. An assortment of mouse models have been engineered to explore the tumorigenic potential and malignant lineage bias due to loss of function of DNMT3A in consort with commonly comutated genes in myeloid malignancies, such as Flt3, Nras, Kras, and c-Kit. We employed several tamoxifen-inducible Cre-ERT2 murine model systems to study the effects of constitutively active KrasG12D-driven myeloid leukemia (Kras) development together with heterozygous (3aHet) or homozygous Dnmt3a deletion (3aKO). Due to the rapid generation of diverse nonhematologic tumors appearing after tamoxifen induction, we employed a transplantation model. With pretransplant tamoxifen induction, most Kras mice died quickly of T-cell malignancies regardless of Dnmt3a status. Using posttransplant induction, we observed a dose-dependent effect of DNMT3A depletion that skewed the leukemic phenotype toward a myeloid lineage. Specifically, 64% of 3aKO/Kras mice had exclusively myeloid disease compared with 36% of 3aHet/Kras and only 13% of Kras mice. Here, 3aKO combined with Kras led to increased disease burden, multiorgan infiltration, and faster disease progression. DOT1L inhibition exerted profound antileukemic effects in malignant 3aKO/Kras cells, but not malignant cells with Kras mutation alone, consistent with the known sensitivity of DNMT3A-mutant leukemia to DOT1L inhibition. RNAseq from malignant myeloid cells revealed that biallelic Dnmt3a deletion was associated with loss of cell-cycle regulation, MYC activation, and TNF⍺ signaling. Overall, we developed a robust model system for mechanistic and preclinical investigations of acute myeloid leukemia with DNMT3A and Ras-pathway lesions.

Human embryonic genetic mosaicism and its effects on development and disease.

Nearly every mammalian cell division is accompanied by a mutational event that becomes fixed in a daughter cell. When carried forward to additional cell progeny, a clone of variant cells can emerge. As a result, mammals are complex mosaics of clones that are genetically distinct from one another. Recent high-throughput sequencing studies have revealed that mosaicism is common, clone sizes often increase with age and specific variants can affect tissue function and disease development. Variants that are acquired during early embryogenesis are shared by multiple cell types and can affect numerous tissues. Within tissues, variant clones compete, which can result in their expansion or elimination. Embryonic mosaicism has clinical implications for genetic disease severity and transmission but is likely an under-recognized phenomenon. To better understand its implications for mosaic individuals, it is essential to leverage research tools that can elucidate the mechanisms by which expanded embryonic variants influence development and disease.

Tissue mosaicism following stem cell aging: blood as an exemplar.

Loss of stem cell regenerative potential underlies aging of all tissues. Somatic mosaicism, the emergence of cellular patchworks within tissues, increases with age and has been observed in every organ yet examined. In the hematopoietic system, as in most tissues, stem cell aging through a variety of mechanisms occurs in lockstep with the emergence of somatic mosaicism. Here, we draw on insights from aging hematopoiesis to illustrate fundamental principles of stem cell aging and somatic mosaicism. We describe the generalizable changes intrinsic to aged stem cells and their milieu that provide the backdrop for somatic mosaicism to emerge. We discuss genetic and nongenetic mechanisms that can result in tissue somatic mosaicism and existing methodologies to detect such clonal outgrowths. Finally, we propose potential avenues to modify mosaicism during aging, with the ultimate aim of increasing tissue resiliency.

Cyclophilin A supports translation of intrinsically disordered proteins and affects haematopoietic stem cell ageing.

Loss of protein function is a driving force of ageing. We have identified peptidyl-prolyl isomerase A (PPIA or cyclophilin A) as a dominant chaperone in haematopoietic stem and progenitor cells. Depletion of PPIA accelerates stem cell ageing. We found that proteins with intrinsically disordered regions (IDRs) are frequent PPIA substrates. IDRs facilitate interactions with other proteins or nucleic acids and can trigger liquid-liquid phase separation. Over 20% of PPIA substrates are involved in the formation of supramolecular membrane-less organelles. PPIA affects regulators of stress granules (PABPC1), P-bodies (DDX6) and nucleoli (NPM1) to promote phase separation and increase cellular stress resistance. Haematopoietic stem cell ageing is associated with a post-transcriptional decrease in PPIA expression and reduced translation of IDR-rich proteins. Here we link the chaperone PPIA to the synthesis of intrinsically disordered proteins, which indicates that impaired protein interaction networks and macromolecular condensation may be potential determinants of haematopoietic stem cell ageing.

Hematologic DNMT3A reduction and high-fat diet synergize to promote weight gain and tissue inflammation.

During aging, blood cell production becomes dominated by a limited number of variant hematopoietic stem cell (HSC) clones. Differentiated progeny of variant HSCs are thought to mediate the detrimental effects of such clonal hematopoiesis on organismal health, but the mechanisms are poorly understood. While somatic mutations in DNA methyltransferase 3A (DNMT3A) frequently drive clonal dominance, the aging milieu also likely contributes. Here, we examined in mice the interaction between high-fat diet (HFD) and reduced DNMT3A in hematopoietic cells; strikingly, this combination led to weight gain. HFD amplified pro-inflammatory pathways and upregulated inflammation-associated genes in mutant cells along a pro-myeloid trajectory. Aberrant DNA methylation during myeloid differentiation and in response to HFD led to pro-inflammatory activation and maintenance of stemness genes. These findings suggest that reduced DNMT3A in hematopoietic cells contributes to weight gain, inflammation, and metabolic dysfunction, highlighting a role for DNMT3A loss in the development of metabolic disorders.

Incipient clonal hematopoiesis is accelerated following CD30.CAR-T therapy.

Chimeric antigen receptor (CAR) T-cells are an emerging therapy for refractory lymphomas. Clonal hematopoiesis (CH), the preferential outgrowth of mutated bone marrow progenitors, is enriched in lymphoma patients receiving CAR-T cells. CAR-T therapy requires conditioning chemotherapy and often induces systemic inflammatory reactions, both of which have been shown to promote expansion of CH clones. Thus, we hypothesized that pre-existing CH clones could expand during CAR-T cell treatment. We measured CH at 154 timepoints longitudinally sampled from 26 patients receiving CD30.CAR-T therapy for CD30+ lymphomas on an investigational protocol (NCT02917083). Pre-treatment CH was present in 54% of individuals and did not correlate with survival outcomes or inflammatory toxicities. Longitudinal tracking of single clones in individual patients revealed distinct clone growth dynamics. Initially small clones, defined as VAF <1%, expanded following CAR-T administration, compared with relatively muted expansions of larger clones (3.37-fold vs. 1.20-fold, P = 0.0014). Matched clones were present at low magnitude in the infused CD30.CAR-T product for all CH cases but did not affect the product's immunophenotype or transduction efficiency. As cellular immunotherapies expand to become frontline treatments for hematological malignancies, our data indicates CAR-T recipients could be enriched for CH, and further longitudinal studies centered on CH complications in this population are warranted.