PURE-seq identifies Egr1 as a Potential Master Regulator in Murine Aging by Sequencing Long-Term Hematopoietic Stem Cells.

Single-cell transcriptomics is valuable for uncovering individual cell properties, particularly in highly heterogeneous systems. However, this technique often results in the analysis of many well-characterized cells, increasing costs and diluting rare cell populations. To address this, we developed PURE-seq (PIP-seq for Rare-cell Enrichment and Sequencing) for scalable sequencing of rare cells. PURE-seq allows direct cell loading from FACS into PIP-seq reactions, minimizing handling and reducing cell loss. PURE-seq reliably captures rare cells, with 60 minutes of sorting capturing tens of cells at a rarity of 1 in 1,000,000. Using PURE-seq, we investigated murine long-term hematopoietic stem cells and their transcriptomes in the context of hematopoietic aging, identifying Egr1 as a potential master regulator of hematopoiesis in the aging context. PURE-seq offers an accessible and reliable method for isolating and sequencing cells that are currently too rare to capture successfully with existing methods.

Beat-AML 2024 ELN-Refined Risk Stratification for Older Adults with Newly Diagnosed AML Given Lower-Intensity Therapy.

While the 2022 European LeukemiaNet (ELN) acute myeloid leukemia (AML) risk classification reliably predicts outcomes in younger patients treated with intensive chemotherapy, it is unclear whether it applies to adults ≥ 60 years treated with lower-intensity treatment (LIT). We aimed to test the prognostic impact of ELN risk in patients with newly diagnosed (ND) AML ≥ 60 years given LIT and to further refine risk stratification for these patients. A total of 595 patients were included: 11% had favorable-risk, 11% had intermediate-risk, and 78% had adverse-risk AML as defined by ELN. ELN risk was prognostic for overall survival (OS) (P<0.001) but did not stratify favorable-risk from intermediate-risk (P=0.71). Within adverse-risk AML, the impact of additional molecular abnormalities was further evaluated. Multivariable analysis was performed on a training set (N=316) and identified IDH2 mutation as an independent favorable prognostic factor, and KRAS, MLL2, and TP53 mutations as unfavorable (P<0.05). A "mutation-score" was calculated for each combination of these mutations, assigning adverse-risk patients into two risk groups: -1 to 0 points ("Beat-AML-intermediate") vs 1+ points ("Beat-AML-adverse"). In the final refined risk classification, the ELN favorable- and intermediate-risk groups were combined into a newly defined "Beat-AML-favorable-risk", in addition to mutation scoring within the ELN adverse-risk. This approach redefines risk for older ND AML and proposes refined Beat-AML-favorable- (22%), Beat-AML-intermediate- (41%), and Beat-AML-adverse-risk (37%) groups with improved discrimination for OS (2-year OS: 48% vs 33% vs 11%, respectively, P<0.001; C-index: 0.60 vs 0.55 for ELN), providing patients and providers additional information for treatment decision-making.

Genetic risk classification for adults with AML receiving less-intensive therapies: the 2024 ELN recommendations.

The European LeukemiaNet (ELN) genetic risk classifications were developed based on data from younger adults receiving intensive chemotherapy. Emerging analyses from patients receiving less-intensive therapies prompted a proposal for an ELN genetic risk classification specifically for this patient population.

Exercise and nutrition to improve cancer Treatment-Related outcomes (ENICTO).

Chemotherapy treatment-related side-effects are common and increase the risk of suboptimal outcomes. Exercise interventions during cancer treatment improve self-reported physical functioning, fatigue, anxiety, and depression, but it is unclear whether these interventions improve important clinical outcomes, such as chemotherapy relative dose intensity (RDI). The National Cancer Institute funded the Exercise and Nutrition to Improve Cancer Treatment-Related Outcomes (ENICTO) Consortium, to address this knowledge gap. This paper describes the mechanisms hypothesized to underpin intervention effects on clinically-relevant treatment outcomes, briefly outlines each project's distinct research aims, summarizes the scope and organizational structure of ENICTO, and provides an overview of the integrated common data elements used to pursue research questions collectively. In addition, the paper includes a description of consortium-wide activities and broader research community opportunities for collaborative research. Findings from the ENICTO Consortium have the potential to accelerate a paradigm shift in oncology care such that cancer patients could receive exercise and nutrition programming as the standard of care in tandem with chemotherapy to improve RDI for a curative outcome.

PURE-seq identifies Egr1 as a Potential Master Regulator in Murine Aging by Sequencing Long-Term Hematopoietic Stem Cells.

Single-cell transcriptomics is valuable for uncovering individual cell properties, particularly in highly heterogeneous systems. However, this technique often results in the analysis of many well-characterized cells, increasing costs and diluting rare cell populations. To address this, we developed PURE-seq (PIP-seq for Rare-cell Enrichment and Sequencing) for scalable sequencing of rare cells. PURE-seq allows direct cell loading from FACS into PIP-seq reactions, minimizing handling and reducing cell loss. PURE-seq reliably captures rare cells, with 60 minutes of sorting capturing tens of cells at a rarity of 1 in 1,000,000. Using PURE-seq, we investigated murine long-term hematopoietic stem cells and their transcriptomes in the context of hematopoietic aging, identifying Egr1 as a potential master regulator of hematopoiesis in the aging context. PURE-seq offers an accessible and reliable method for isolating and sequencing cells that are currently too rare to capture successfully with existing methods.

Developing a membrane-proximal CD33-targeting CAR T cell.

BACKGROUND: CD33 is a tractable target in acute myeloid leukemia (AML) for chimeric antigen receptor (CAR) T cell therapy, but clinical success is lacking. METHODS: We developed 3P14HLh28Z, a novel CD33-directed CD28/CD3Z-based CAR T cell derived from a high-affinity binder obtained through membrane-proximal fragment immunization in humanized mice. RESULTS: We found that immunization exclusively with the membrane-proximal domain of CD33 is necessary for identification of membrane-proximal binders in humanized mice. Compared with clinically validated lintuzumab-based CAR T cells targeting distal CD33 epitopes, 3P14HLh28Z showed enhanced in vitro functionality as well as superior tumor control and increased overall survival in both low antigen density and clinically relevant patient-derived xenograft models. Increased activation and enhanced polyfunctionality led to enhanced efficacy. CONCLUSIONS: Showing for the first time that a membrane-proximal CAR is superior to a membrane-distal one in the setting of CD33 targeting, our results demonstrate the rationale for targeting membrane-proximal epitopes with high-affinity binders. We also demonstrate the importance of optimizing CAR T cells for functionality in settings of both low antigen density and clinically relevant patient-derived models.

CRISPR Dependency Screens in Primary Hematopoietic Stem Cells Identify KDM3B as a Genotype Specific Vulnerability in IDH2- and TET2-Mutant Cells.

Clonal hematopoiesis (CH) is a common premalignant state in the blood and confers an increased risk of blood cancers and all-cause mortality. Identification of therapeutic targets in CH has been hindered by the lack of an ex vivo platform amenable for studying primary hematopoietic stem and progenitor cells (HSPCs). Here, we utilize an ex vivo co-culture system of HSPCs with bone marrow endothelial cells to perform CRISPR/Cas9 screens in mutant HSPCs. Our data reveal that loss of the histone demethylase family members Kdm3b and Jmjd1c specifically reduces the fitness of Idh2- and Tet2-mutant HSPCs. Kdm3b loss in mutant cells leads to decreased expression of critical cytokine receptors including Mpl, rendering mutant HSPCs preferentially susceptible to inhibition of downstream JAK2 signaling. Our study nominates an epigenetic regulator and an epigenetically regulated receptor signaling pathway as genotype-specific therapeutic targets and provides a scalable platform to identify genetic dependencies in mutant HSPCs.

Mapping genotypes to chromatin accessibility profiles in single cells.

In somatic tissue differentiation, chromatin accessibility changes govern priming and precursor commitment towards cellular fates1-3. Therefore, somatic mutations are likely to alter chromatin accessibility patterns, as they disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wild-type cells. Here, to chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed genotyping of targeted loci with single-cell chromatin accessibility (GoT-ChA). This high-throughput platform links genotypes to chromatin accessibility at single-cell resolution across thousands of cells within a single assay. We applied GoT-ChA to CD34+ cells from patients with myeloproliferative neoplasms with JAK2V617F-mutated haematopoiesis. Differential accessibility analysis between wild-type and JAK2V617F-mutant progenitors revealed both cell-intrinsic and cell-state-specific shifts within mutant haematopoietic precursors, including cell-intrinsic pro-inflammatory signatures in haematopoietic stem cells, and a distinct profibrotic inflammatory chromatin landscape in megakaryocytic progenitors. Integration of mitochondrial genome profiling and cell-surface protein expression measurement allowed expansion of genotyping onto DOGMA-seq through imputation, enabling single-cell capture of genotypes, chromatin accessibility, RNA expression and cell-surface protein expression. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner, influencing inflammation states and differentiation trajectories. We envision that GoT-ChA will empower broad future investigations of the critical link between somatic mutations and epigenetic alterations across clonal populations in malignant and non-malignant contexts.

Development of a customizable mouse backbone spectral flow cytometry panel to delineate immune cell populations in normal and tumor tissues.

Introduction: In vivo studies of cancer biology and assessment of therapeutic efficacy are critical to advancing cancer research and ultimately improving patient outcomes. Murine cancer models have proven to be an invaluable tool in pre-clinical studies. In this context, multi-parameter flow cytometry is a powerful method for elucidating the profile of immune cells within the tumor microenvironment and/or play a role in hematological diseases. However, designing an appropriate multi-parameter panel to comprehensively profile the increasing diversity of immune cells across different murine tissues can be extremely challenging. Methods: To address this issue, we designed a panel with 13 fixed markers that define the major immune populations -referred to as the backbone panel- that can be profiled in different tissues but with the option to incorporate up to seven additional fluorochromes, including any marker specific to the study in question. Results: This backbone panel maintains its resolution across different spectral flow cytometers and organs, both hematopoietic and non-hematopoietic, as well as tumors with complex immune microenvironments. Discussion: Having a robust backbone that can be easily customized with pre-validated drop-in fluorochromes saves time and resources and brings consistency and standardization, making it a versatile solution for immuno-oncology researchers. In addition, the approach presented here can serve as a guide to develop similar types of customizable backbone panels for different research questions requiring high-parameter flow cytometry panels.

Mesenchymal Stromal Cell Senescence Induced by Dnmt3a -Mutant Hematopoietic Cells is a Targetable Mechanism Driving Clonal Hematopoiesis and Initiation of Hematologic Malignancy.

Clonal hematopoiesis (CH) can predispose to blood cancers due to enhanced fitness of mutant hematopoietic stem and progenitor cells (HSPCs), but the mechanisms driving this progression are not understood. We hypothesized that malignant progression is related to microenvironment-remodelling properties of CH-mutant HSPCs. Single-cell transcriptomic profiling of the bone marrow microenvironment in Dnmt3a R878H/+ mice revealed signatures of cellular senescence in mesenchymal stromal cells (MSCs). Dnmt3a R878H/+ HSPCs caused MSCs to upregulate the senescence markers SA-ß-gal, BCL-2, BCL-xL, Cdkn1a (p21) and Cdkn2a (p16), ex vivo and in vivo . This effect was cell contact-independent and can be replicated by IL-6 or TNFα, which are produced by Dnmt3a R878H/+ HSPCs. Depletion of senescent MSCs in vivo reduced the fitness of Dnmt3a R878H/+ hematopoietic cells and the progression of CH to myeloid neoplasms using a sequentially inducible Dnmt3a ; Npm1 -mutant model. Thus, Dnmt3a -mutant HSPCs reprogram their microenvironment via senescence induction, creating a self-reinforcing niche favoring fitness and malignant progression. Statement of Significance: Mesenchymal stromal cell senescence induced by Dnmt3a -mutant hematopoietic stem and progenitor cells drives clonal hematopoiesis and initiation of hematologic malignancy.

Mutation order in acute myeloid leukemia identifies uncommon patterns of evolution and illuminates phenotypic heterogeneity.

Acute myeloid leukemia (AML) has a poor prognosis and a heterogeneous mutation landscape. Although common mutations are well-studied, little research has characterized how the sequence of mutations relates to clinical features. Using published, single-cell DNA sequencing data from three institutions, we compared clonal evolution patterns in AML to patient characteristics, disease phenotype, and outcomes. Mutation trees, which represent the order of select mutations, were created for 207 patients from targeted panel sequencing data using 1 639 162 cells, 823 mutations, and 275 samples. In 224 distinct orderings of mutated genes, mutations related to DNA methylation typically preceded those related to cell signaling, but signaling-first cases did occur, and had higher peripheral cell counts, increased signaling mutation homozygosity, and younger patient age. Serial sample analysis suggested that NPM1 and DNA methylation mutations provide an advantage to signaling mutations in AML. Interestingly, WT1 mutation evolution shared features with signaling mutations, such as WT1-early being proliferative and occurring in younger individuals, trends that remained in multivariable regression. Some mutation orderings had a worse prognosis, but this was mediated by unfavorable mutations, not mutation order. These findings add a dimension to the mutation landscape of AML, identifying uncommon patterns of leukemogenesis and shedding light on heterogeneous phenotypes.

Suppression of IL-1ß promotes beneficial accumulation of fibroblast-like cells in atherosclerotic plaques in clonal hematopoiesis.

Clonal hematopoiesis (CH) is an independent risk factor for atherosclerotic cardiovascular disease. Murine models of CH suggest a central role of inflammasomes and IL-1ß in accelerated atherosclerosis and plaque destabilization. Here we show using single-cell RNA sequencing in human carotid plaques that inflammasome components are enriched in macrophages, while the receptor for IL-1ß is enriched in fibroblasts and smooth muscle cells (SMCs). To address the role of inflammatory crosstalk in features of plaque destabilization, we conducted SMC fate mapping in Ldlr-/- mice modeling Jak2VF or Tet2 CH treated with IL-1ß antibodies. Unexpectedly, this treatment minimally affected SMC differentiation, leading instead to a prominent expansion of fibroblast-like cells. Depletion of fibroblasts from mice treated with IL-1ß antibody resulted in thinner fibrous caps. Conversely, genetic inactivation of Jak2VF during plaque regression promoted fibroblast accumulation and fibrous cap thickening. Our studies suggest that suppression of inflammasomes promotes plaque stabilization by recruiting fibroblast-like cells to the fibrous cap.

Jak2V617F Reversible Activation Shows Its Essential Requirement in Myeloproliferative Neoplasms.

Gain-of-function mutations activating JAK/STAT signaling are seen in the majority of patients with myeloproliferative neoplasms (MPN), most commonly JAK2V617F. Although clinically approved JAK inhibitors improve symptoms and outcomes in MPNs, remissions are rare, and mutant allele burden does not substantively change with chronic therapy. We hypothesized this is due to limitations of current JAK inhibitors to potently and specifically abrogate mutant JAK2 signaling. We therefore developed a conditionally inducible mouse model allowing for sequential activation, and then inactivation, of Jak2V617F from its endogenous locus using a combined Dre-rox/Cre-lox dual-recombinase system. Jak2V617F deletion abrogates MPN features, induces depletion of mutant-specific hematopoietic stem/progenitor cells, and extends overall survival to an extent not observed with pharmacologic JAK inhibition, including when cooccurring with somatic Tet2 loss. Our data suggest JAK2V617F represents the best therapeutic target in MPNs and demonstrate the therapeutic relevance of a dual-recombinase system to assess mutant-specific oncogenic dependencies in vivo. SIGNIFICANCE: Current JAK inhibitors to treat myeloproliferative neoplasms are ineffective at eradicating mutant cells. We developed an endogenously expressed Jak2V617F dual-recombinase knock-in/knock-out model to investigate Jak2V617F oncogenic reversion in vivo. Jak2V617F deletion abrogates MPN features and depletes disease-sustaining MPN stem cells, suggesting improved Jak2V617F targeting offers the potential for greater therapeutic efficacy. See related commentary by Celik and Challen, p. 701. This article is featured in Selected Articles from This Issue, p. 695.

FLT3 tyrosine kinase inhibition modulates PRC2 and promotes differentiation in acute myeloid leukemia.

Internal tandem duplication mutations in fms-like tyrosine kinase 3 (FLT3-ITD) are recurrent in acute myeloid leukemia (AML) and increase the risk of relapse. Clinical responses to FLT3 inhibitors (FLT3i) include myeloid differentiation of the FLT3-ITD clone in nearly half of patients through an unknown mechanism. We identified enhancer of zeste homolog 2 (EZH2), a component of polycomb repressive complex 2 (PRC2), as a mediator of this effect using a proteomic-based screen. FLT3i downregulated EZH2 protein expression and PRC2 activity on H3K27me3. FLT3-ITD and loss-of-function mutations in EZH2 are mutually exclusive in human AML. We demonstrated that FLT3i increase myeloid maturation with reduced stem/progenitor cell populations in murine Flt3-ITD AML. Combining EZH1/2 inhibitors with FLT3i increased terminal maturation of leukemic cells and reduced leukemic burden. Our data suggest that reduced EZH2 activity following FLT3 inhibition promotes myeloid differentiation of FLT3-ITD leukemic cells, providing a mechanistic explanation for the clinical observations. These results demonstrate that in addition to its known cell survival and proliferation signaling, FLT3-ITD has a second, previously undefined function to maintain a myeloid stem/progenitor cell state through modulation of PRC2 activity. Our findings support exploring EZH1/2 inhibitors as therapy for FLT3-ITD AML.

Prophylactic and long-lasting efficacy of senolytic CAR T cells against age-related metabolic dysfunction.

Senescent cells, which accumulate in organisms over time, contribute to age-related tissue decline. Genetic ablation of senescent cells can ameliorate various age-related pathologies, including metabolic dysfunction and decreased physical fitness. While small-molecule drugs that eliminate senescent cells ('senolytics') partially replicate these phenotypes, they require continuous administration. We have developed a senolytic therapy based on chimeric antigen receptor (CAR) T cells targeting the senescence-associated protein urokinase plasminogen activator receptor (uPAR), and we previously showed these can safely eliminate senescent cells in young animals. We now show that uPAR-positive senescent cells accumulate during aging and that they can be safely targeted with senolytic CAR T cells. Treatment with anti-uPAR CAR T cells improves exercise capacity in physiological aging, and it ameliorates metabolic dysfunction (for example, improving glucose tolerance) in aged mice and in mice on a high-fat diet. Importantly, a single administration of these senolytic CAR T cells is sufficient to achieve long-term therapeutic and preventive effects.

Quantifying the Expanding Landscape of Clinical Actionability for Patients with Cancer.

There is a continuing debate about the proportion of cancer patients that benefit from precision oncology, attributable in part to conflicting views as to which molecular alterations are clinically actionable. To quantify the expansion of clinical actionability since 2017, we annotated 47,271 solid tumors sequenced with the MSK-IMPACT clinical assay using two temporally distinct versions of the OncoKB knowledge base deployed 5 years apart. Between 2017 and 2022, we observed an increase from 8.9% to 31.6% in the fraction of tumors harboring a standard care (level 1 or 2) predictive biomarker of therapy response and an almost halving of tumors carrying nonactionable drivers (44.2% to 22.8%). In tumors with limited or no clinical actionability, TP53 (43.2%), KRAS (19.2%), and CDKN2A (12.2%) were the most frequently altered genes. SIGNIFICANCE: Although clear progress has been made in expanding the availability of precision oncology-based treatment paradigms, our results suggest a continued unmet need for innovative therapeutic strategies, particularly for cancers with currently undruggable oncogenic drivers. See related commentary by Horak and Fröhling, p. 18. This article is featured in Selected Articles from This Issue, p. 5.

Development of Resistance to Type II JAK2 Inhibitors in MPN Depends on AXL Kinase and Is Targetable.

PURPOSE: Myeloproliferative neoplasms (MPN) dysregulate JAK2 signaling. Because clinical JAK2 inhibitors have limited disease-modifying effects, type II JAK2 inhibitors such as CHZ868 stabilizing inactive JAK2 and reducing MPN clones, gain interest. We studied whether MPN cells escape from type ll inhibition. EXPERIMENTAL DESIGN: MPN cells were continuously exposed to CHZ868. We used phosphoproteomic analyses and ATAC/RNA sequencing to characterize acquired resistance to type II JAK2 inhibition, and targeted candidate mediators in MPN cells and mice. RESULTS: MPN cells showed increased IC50 and reduced apoptosis upon CHZ868 reflecting acquired resistance to JAK2 inhibition. Among >2,500 differential phospho-sites, MAPK pathway activation was most prominent, while JAK2-STAT3/5 remained suppressed. Altered histone occupancy promoting AP-1/GATA binding motif exposure associated with upregulated AXL kinase and enriched RAS target gene profiles. AXL knockdown resensitized MPN cells and combined JAK2/AXL inhibition using bemcentinib or gilteritinib reduced IC50 to levels of sensitive cells. While resistant cells induced tumor growth in NOD/SCID gamma mice despite JAK2 inhibition, JAK2/AXL inhibition largely prevented tumor progression. Because inhibitors of MAPK pathway kinases such as MEK are clinically used in other malignancies, we evaluated JAK2/MAPK inhibition with trametinib to interfere with AXL/MAPK-induced resistance. Tumor growth was halted similarly to JAK2/AXL inhibition and in a systemic cell line-derived mouse model, marrow infiltration was decreased supporting dependency on AXL/MAPK. CONCLUSIONS: We report on a novel mechanism of AXL/MAPK-driven escape from type II JAK2 inhibition, which is targetable at different nodes. This highlights AXL as mediator of acquired resistance warranting inhibition to enhance sustainability of JAK2 inhibition in MPN.