Single-cell analyses of metastatic bone marrow in human neuroblastoma reveals microenvironmental remodeling and metastatic signature.

Neuroblastoma is an aggressive pediatric cancer with a high rate of metastasis to the BM. Despite intensive treatments including high-dose chemotherapy, the overall survival rate for children with metastatic neuroblastoma remains dismal. Understanding the cellular and molecular mechanisms of the metastatic tumor microenvironment is crucial for developing new therapies and improving clinical outcomes. Here, we used single-cell RNA-Seq to characterize immune and tumor cell alterations in neuroblastoma BM metastases by comparative analysis with patients without metastases. Our results reveal remodeling of the immune cell populations and reprogramming of gene expression profiles in the metastatic niche. In particular, within the BM metastatic niche, we observed the enrichment of immune cells, including tumor-associated neutrophils, macrophages, and exhausted T cells, as well as an increased number of Tregs and a decreased number of B cells. Furthermore, we highlighted cell communication between tumor cells and immune cell populations, and we identified prognostic markers in malignant cells that are associated with worse clinical outcomes in 3 independent neuroblastoma cohorts. Our results provide insight into the cellular, compositional, and transcriptional shifts underlying neuroblastoma BM metastases that contribute to the development of new therapeutic strategies.

Integrative analysis of neuroblastoma by single-cell RNA sequencing identifies the NECTIN2-TIGIT axis as a target for immunotherapy.

Pediatric patients with high-risk neuroblastoma have poor survival rates and urgently need more effective treatment options with less side effects. Since novel and improved immunotherapies may fill this need, we dissect the immunoregulatory interactions in neuroblastoma by single-cell RNA-sequencing of 24 tumors (10 pre- and 14 post-chemotherapy, including 5 pairs) to identify strategies for optimizing immunotherapy efficacy. Neuroblastomas are infiltrated by natural killer (NK), T and B cells, and immunosuppressive myeloid populations. NK cells show reduced cytotoxicity and T cells have a dysfunctional profile. Interaction analysis reveals a vast immunoregulatory network and identifies NECTIN2-TIGIT as a crucial immune checkpoint. Combined blockade of TIGIT and PD-L1 significantly reduces neuroblastoma growth, with complete responses (CR) in vivo. Moreover, addition of TIGIT+PD-L1 blockade to standard relapse treatment in a chemotherapy-resistant Th-ALKF1174L/MYCN 129/SvJ syngeneic model induces CR. In conclusion, our integrative analysis provides promising targets and a rationale for immunotherapeutic combination strategies.

Single-cell analysis of immune and stroma cell remodeling in clear cell renal cell carcinoma primary tumors and bone metastatic lesions.

BACKGROUND: Despite therapeutic advances, once a cancer has metastasized to the bone, it represents a highly morbid and lethal disease. One third of patients with advanced clear cell renal cell carcinoma (ccRCC) present with bone metastasis at the time of diagnosis. However, the bone metastatic niche in humans, including the immune and stromal microenvironments, has not been well-defined, hindering progress towards identification of therapeutic targets. METHODS: We collected fresh patient samples and performed single-cell transcriptomic profiling of solid metastatic tissue (Bone Met), liquid bone marrow at the vertebral level of spinal cord compression (Involved), and liquid bone marrow from a different vertebral body distant from the tumor site but within the surgical field (Distal), as well as bone marrow from patients undergoing hip replacement surgery (Benign). In addition, we incorporated single-cell data from primary ccRCC tumors (ccRCC Primary) for comparative analysis. RESULTS: The bone marrow of metastatic patients is immune-suppressive, featuring increased, exhausted CD8 + cytotoxic T cells, T regulatory cells, and tumor-associated macrophages (TAM) with distinct transcriptional states in metastatic lesions. Bone marrow stroma from tumor samples demonstrated a tumor-associated mesenchymal stromal cell population (TA-MSC) that appears to be supportive of epithelial-to mesenchymal transition (EMT), bone remodeling, and a cancer-associated fibroblast (CAFs) phenotype. This stromal subset is associated with poor progression-free and overall survival and also markedly upregulates bone remodeling through the dysregulation of RANK/RANKL/OPG signaling activity in bone cells, ultimately leading to bone resorption. CONCLUSIONS: These results provide a comprehensive analysis of the bone marrow niche in the setting of human metastatic cancer and highlight potential therapeutic targets for both cell populations and communication channels.

Recent Advances in Single-Cell RNA-Sequencing of Primary and Metastatic Clear Cell Renal Cell Carcinoma.

Over the past two decades, significant progress has been made in the treatment of clear cell renal cell carcinoma (ccRCC), with a shift towards adopting new treatment approaches ranging from monotherapy to triple-combination therapy. This progress has been spearheaded by fundamental technological advancements that have allowed a deeper understanding of the various biological components of this cancer. In particular, the rapid commercialization of transcriptomics technologies, such as single-cell RNA-sequencing (scRNA-seq) methodologies, has played a crucial role in accelerating this understanding. Through precise measurements facilitated by these technologies, the research community has successfully identified and characterized diverse tumor, immune, and stromal cell populations, uncovering their interactions and pathways involved in disease progression. In localized ccRCC, patients have shown impressive response rates to treatment. However, despite the emerging findings and new knowledge provided in the field, there are still patients that do not respond to treatment, especially in advanced disease stages. One of the key challenges lies in the limited study of ccRCC metastases compared to localized cases. This knowledge gap may contribute to the relatively low survival rates and response rates observed in patients with metastatic ccRCC. To bridge this gap, we here delve into recent research utilizing scRNA-seq technologies in both primary and metastatic ccRCC. The goal of this review is to shed light on the current state of knowledge in the field, present existing treatment options, and emphasize the crucial steps needed to improve survival rates, particularly in cases of metastatic ccRCC.

Adult Prg4+ progenitors repair long-term articular cartilage wounds in vivo.

The identity and origin of the stem/progenitor cells for adult joint cartilage repair remain unknown, impeding therapeutic development. Simulating the common therapeutic modality for cartilage repair in humans, i.e., full-thickness microfracture joint surgery, we combined the mouse full-thickness injury model with lineage tracing and identified a distinct skeletal progenitor cell type enabling long-term (beyond 7 days after injury) articular cartilage repair in vivo. Deriving from a population with active Prg4 expression in adulthood while lacking aggrecan expression, these progenitors proliferate, differentiate to express aggrecan and type II collagen, and predominate in long-term articular cartilage wounds, where they represent the principal repair progenitors in situ under native repair conditions without cellular transplantation. They originate outside the adult bone marrow or superficial zone articular cartilage. These findings have implications for skeletal biology and regenerative medicine for joint injury repair.

Macrophage infiltration promotes regrowth in MYCN-amplified neuroblastoma after chemotherapy.

Despite aggressive treatment, the 5-year event-free survival rate for children with high-risk neuroblastoma is <50%. While most high-risk neuroblastoma patients initially respond to treatment, often with complete clinical remission, many eventually relapse with therapy-resistant tumors. Novel therapeutic alternatives that prevent the recurrence of therapy-resistant tumors are urgently needed. To understand the adaptation of neuroblastoma under therapy, we analyzed the transcriptomic landscape in 46 clinical tumor samples collected before (PRE) or after (POST) treatment from 22 neuroblastoma patients. RNA sequencing revealed that many of the top-upregulated biological processes in POST MYCN amplified (MNA+) tumors compared to PRE MNA+ tumors were immune-related, and there was a significant increase in numerous genes associated with macrophages. The infiltration of macrophages was corroborated by immunohistochemistry and spatial digital protein profiling. Moreover, POST MNA+ tumor cells were more immunogenic compared to PRE MNA+ tumor cells. To find support for the macrophage-induced outgrowth of certain subpopulations of immunogenic tumor cells following treatment, we examined the genetic landscape in multiple clinical PRE and POST tumor samples from nine neuroblastoma patients revealing a significant correlation between an increased amount of copy number aberrations (CNA) and macrophage infiltration in POST MNA+ tumor samples. Using an in vivo neuroblastoma patient-derived xenograft (PDX) chemotherapy model, we further show that inhibition of macrophage recruitment with anti-CSF1R treatment prevents the regrowth of MNA+ tumors following chemotherapy. Taken together, our work supports a therapeutic strategy for fighting the relapse of MNA+ neuroblastoma by targeting the immune microenvironment.

Dissecting the immune suppressive human prostate tumor microenvironment via integrated single-cell and spatial transcriptomic analyses.

The treatment of low-risk primary prostate cancer entails active surveillance only, while high-risk disease requires multimodal treatment including surgery, radiation therapy, and hormonal therapy. Recurrence and development of metastatic disease remains a clinical problem, without a clear understanding of what drives immune escape and tumor progression. Here, we comprehensively describe the tumor microenvironment of localized prostate cancer in comparison with adjacent normal samples and healthy controls. Single-cell RNA sequencing and high-resolution spatial transcriptomic analyses reveal tumor context dependent changes in gene expression. Our data indicate that an immune suppressive tumor microenvironment associates with suppressive myeloid populations and exhausted T-cells, in addition to high stromal angiogenic activity. We infer cell-to-cell relationships from high throughput ligand-receptor interaction measurements within undissociated tissue sections. Our work thus provides a highly detailed and comprehensive resource of the prostate tumor microenvironment as well as tumor-stromal cell interactions.

Role of reactive oxygen species in regulating 27-hydroxycholesterol-induced apoptosis of hematopoietic progenitor cells and myeloid cell lines.

Oxysterols are oxygenated derivatives of cholesterol that contain an additional hydroxy, epoxide, or ketone group in the sterol nucleus and/or a hydroxyl group in the side chain of the cholesterol molecule. 27-Hydroxycholesterol (27HC) is a side-chain oxysterol that is oxygenated at the 27th carbon atom of cholesterol. The oxysterol (27HC) is produced via oxidation by sterol 27-hydroxylase (CYP27A1) and metabolized via oxysterol 7a-hydroxylase (CYP7B1) for bile acid synthesis in the liver. A previous study has demonstrated that treatment with the alternative Estrogen receptor alpha (ERα) ligand 27HC induces ERα-dependent hematopoietic stem cell (HSC) mobilization. In addition, Cyp27a1-deficient mice demonstrate significantly reduced 27HC levels and HSC mobilization. Here, we report that exogenous 27HC treatment leads to a substantial reduction in the hematopoietic stem and progenitor cell (HSPC) population owing to significantly increased reactive oxygen species (ROS) levels and apoptosis in the bone marrow (BM). However, 27HC does not influence the population of mature hematopoietic cells in the BM. Furthermore, exogenous 27HC treatment suppresses cell growth and promotes ROS production and apoptosis in leukemic cells. Moreover, acute myeloid leukemia (AML) patients with high CYP7B1 expression (expected to have inhibition of 27HC) had significantly shorter survival than those with low CYP7B1 expression (expected to have an elevation of 27HC). Single-cell RNA-sequencing (scRNA seq) analysis revealed that the expression of CYP7B1 was significantly increased in AML patients. Thus, our study suggests that 27HC may serve as a potent agent for regulating pools of HSPCs and may have an application as a novel therapeutic target for hematological malignancies. Collectively, pharmacological inhibition of CYP7B1 (expected to have an elevation of 27HC) would potentially have fewer long-term hematological side effects, particularly when used in combination with chemotherapy or radiation for the treatment of leukemia patients.

A transcriptional metastatic signature predicts survival in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer in adults. When ccRCC is localized to the kidney, surgical resection or ablation of the tumor is often curative. However, in the metastatic setting, ccRCC remains a highly lethal disease. Here we use fresh patient samples that include treatment-naive primary tumor tissue, matched adjacent normal kidney tissue, as well as tumor samples collected from patients with bone metastases. Single-cell transcriptomic analysis of tumor cells from the primary tumors reveals a distinct transcriptional signature that is predictive of metastatic potential and patient survival. Analysis of supporting stromal cells within the tumor environment demonstrates vascular remodeling within the endothelial cells. An in silico cell-to-cell interaction analysis highlights the CXCL9/CXCL10-CXCR3 axis and the CD70-CD27 axis as potential therapeutic targets. Our findings provide biological insights into the interplay between tumor cells and the ccRCC microenvironment.

DHODH is an independent prognostic marker and potent therapeutic target in neuroblastoma.

Despite intensive therapy, children with high-risk neuroblastoma are at risk of treatment failure. We applied a multiomic system approach to evaluate metabolic vulnerabilities in human neuroblastoma. We combined metabolomics, CRISPR screening, and transcriptomic data across more than 700 solid tumor cell lines and identified dihydroorotate dehydrogenase (DHODH), a critical enzyme in pyrimidine synthesis, as a potential treatment target. Of note, DHODH inhibition is currently under clinical investigation in patients with hematologic malignancies. In neuroblastoma, DHODH expression was identified as an independent risk factor for aggressive disease, and high DHODH levels correlated to worse overall and event-free survival. A subset of tumors with the highest DHODH expression was associated with a dismal prognosis, with a 5-year survival of less than 10%. In xenograft and transgenic neuroblastoma mouse models treated with the DHODH inhibitor brequinar, tumor growth was dramatically reduced, and survival was extended. Furthermore, brequinar treatment was shown to reduce the expression of MYC targets in 3 neuroblastoma models in vivo. A combination of brequinar and temozolomide was curative in the majority of transgenic TH-MYCN neuroblastoma mice, indicating a highly active clinical combination therapy. Overall, DHODH inhibition combined with temozolomide has therapeutic potential in neuroblastoma, and we propose this combination for clinical testing.

The immune cell atlas of human neuroblastoma.

Understanding the complete immune cell composition of human neuroblastoma (NB) is crucial for the development of immunotherapeutics. Here, we perform single-cell RNA sequencing (scRNA-seq) on 19 human NB samples coupled with multiplex immunohistochemistry, survival analysis, and comparison with normal fetal adrenal gland data. We provide a comprehensive immune cell landscape and characterize cell-state changes from normal tissue to NB. Our analysis reveals 27 immune cell subtypes, including distinct subpopulations of myeloid, NK, B, and T cells. Several different cell types demonstrate a survival benefit. In contrast to adult cancers and previous NB studies, we show an increase in inflammatory monocyte cell state when contrasting normal and tumor tissue, while no differences in cytotoxicity and exhaustion score for T cells, nor in Treg activity, are observed. Our receptor-ligand interaction analysis reveals a highly complex interactive network of the NB microenvironment from which we highlight several interactions that we suggest for future therapeutic studies.

Oxime derivative TFOBO promotes cell death by modulating reactive oxygen species and regulating NADPH oxidase activity in myeloid leukemia.

Several derivatives derived from the oxime structure have been reported as potential anticancer agents in various cancers. Here, we first tested a novel oxime-containing derivative of 2-((2,4,5-trifluorobenzyl)oxy)benzaldehyde oxime (TFOBO) to evaluate its anticancer effect in myeloid leukemic cells. Compared to (2-((2,4,5-trifluorobenzyl)oxy)phenyl)methanol (TFOPM), the oxime derivative TFOBO suppresses leukemic cell growth by significantly increasing reactive oxygen species (ROS) levels and cell death. Leukemic cells treated with TFOBO displayed apoptotic cell death, as indicated by nuclear condensation, DNA fragmentation, and annexin V staining. TFOBO increases Bax/Bcl2 levels, caspase9, and caspase3/7 activity and decreases mitochondrial membrane potential. ROS production was reduced by N-acetyl-L-cysteine, a ROS scavenger, diphenyleneiodonium chloride, a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, after exogenous TFOBO treatment. ROS inhibitors protect leukemic cells from TFOBO-induced cell death. Thus, our study findings suggest that TFOBO promotes apoptosis by modulating ROS and regulating NADPH oxidase activity. Collectively, the oxime-containing derivative TFOBO is a novel therapeutic drug for myeloid leukemia.

Risk stratification of patients with right-sided colorectal cancer based on the tumor-infiltrating M1 macrophage.

The homing of M1 and M2 macrophages may play distinct roles in the tumor microenvironment (TME). However, these roles of macrophages in the TME remain unclear. We downloaded RNA sequencing data from The Cancer Genome Atlas (TCGA) database for patients with CRC. Subsequently, Kaplan-Meier survival curves were generated to assess the differential infiltration of M1 and M2 macrophages based on CRC location. Differentially expressed gene (DEG) and functional analyses were performed to screen the roles of DEGs. Critical prognostic genes were identified using least absolute shrinkage and selection operator regression. The risk scores were calculated for each patient. In patients with right-sided CRC, reduced M1 macrophage infiltration was associated with poor prognosis. M1 macrophage infiltration positively correlated with CD8+ T cell infiltration. A risk model was developed and validated for performance using GSE103479 and GSE72970. Nine genes were identified as independent prognostic genes that could be potential biomarkers for effectively predicting survival in patients with right-sided CRC. Kaplan-Meier curves for overall survival and progression-free survival analyses revealed that the high-risk group of patients with right-sided CRC had a poor prognosis. This novel M1 macrophage-related risk model may provide a gene signature for predicting the survival outcomes of patients with right-sided CRC and facilitate further studies examining the relationship between infiltration of M1 macrophages and the prognosis of such patients.

External signals regulate continuous transcriptional states in hematopoietic stem cells.

Hematopoietic stem cells (HSCs) must ensure adequate blood cell production following distinct external stressors. A comprehensive understanding of in vivo heterogeneity and specificity of HSC responses to external stimuli is currently lacking. We performed single-cell RNA sequencing (scRNA-Seq) on functionally validated mouse HSCs and LSK (Lin-, c-Kit+, Sca1+) progenitors after in vivo pharmacological perturbation of niche signals interferon, granulocyte colony-stimulating factor (G-CSF), and prostaglandin. We identified six HSC states that are characterized by enrichment but not exclusive expression of marker genes. External signals induced rapid transitions between HSC states but transcriptional response varied both between external stimulants and within the HSC population for a given perturbation. In contrast to LSK progenitors, HSCs were characterized by a greater link between molecular signatures at baseline and in response to external stressors. Chromatin analysis of unperturbed HSCs and LSKs by scATAC-Seq suggested some HSC-specific, cell intrinsic predispositions to niche signals. We compiled a comprehensive resource of HSC- and LSK progenitor-specific chromatin and transcriptional features that represent determinants of signal receptiveness and regenerative potential during stress hematopoiesis.

Most organs in the human body are maintained by a type of immature cells known as adult stem cells, which ensure a constant supply of new, mature cells. Adult stem cells monitor their environment through external signalling molecules and replace damaged cells as needed. Stem cell therapy takes advantage of the regenerative ability of immature stem cells and can be helpful for conditions such as blood diseases, autoimmune diseases, neurodegeneration and cancer. For example, hematopoietic stem-cell transplantation is a treatment for some types of cancer and blood disorders, in which stem cells are harvested from the blood or bone marrow and reintroduced into the body, where they can develop into all types of blood cells, including white blood cells, red blood cells and platelets. Hematopoietic stem-cell transplants have been in use for over 30 years, but they remain a highly risky procedure. One of the challenges is that outcomes can vary between patients and many of the factors that can influence the 'regenerative' potential of hematopoietic stem cells, such as external signalling molecules, are not well understood. To fill this gap, Fast et al. analysed which genes are turned on and off in hematopoietic stem cells in response to several external signalling molecules. To do so, three signalling pathways in mice were altered by injecting them with different chemicals. After two hours, the hematopoietic stem cells were purified and the gene expression for each cell was analysed. This revealed that the types of genes and the strength at which they were affected by each chemical was unique. Moreover, hematopoietic stem cells responded rapidly to external signals, with substantial differences in gene expression between individual groups of cells. Contrary to more specialised cells, the external signalling genes in some hematopoietic stem cells were already activated without being injected with external signalling molecules. This suggest that low levels of external signalling molecules released from their microenvironment may prepare stem cells to better respond to future stress or injuries. These results help to better understand stem cells and to evaluate how the signalling state of hematopoietic stem cells affects regeneration, and ultimately improve hematopoietic stem cell transplantation for patients.

Human prostate cancer bone metastases have an actionable immunosuppressive microenvironment.

Bone metastases are devastating complications of cancer. They are particularly common in prostate cancer (PCa), represent incurable disease, and are refractory to immunotherapy. We seek to define distinct features of the bone marrow (BM) microenvironment by analyzing single cells from bone metastatic prostate tumors, involved BM, uninvolved BM, and BM from cancer-free, orthopedic patients, and healthy individuals. Metastatic PCa is associated with multifaceted immune distortion, specifically exhaustion of distinct T cell subsets, appearance of macrophages with states specific to PCa bone metastases. The chemokine CCL20 is notably overexpressed by myeloid cells, as is its cognate CCR6 receptor on T cells. Disruption of the CCL20-CCR6 axis in mice with syngeneic PCa bone metastases restores T cell reactivity and significantly prolongs animal survival. Comparative high-resolution analysis of PCa bone metastases shows a targeted approach for relieving local immunosuppression for therapeutic effect.

High Expression of PPM1D Induces Tumors Phenotypically Similar to TP53 Loss-of-Function Mutations in Mice.

PPM1D is a negative regulator of p53 and genomic aberrations resulting in increased activity of PPM1D have been observed in cancers of different origins, indicating that PPM1D has oncogenic properties. We established a transgenic mouse model overexpressing PPM1D and showed that these mice developed a wide variety of cancers. PPM1D-expressing mice developed tumors phenotypically and genetically similar to tumors in mice with dysfunctional p53. T-cell lymphoblastic lymphoma was the most frequent cancer observed in these mice (55%) followed by adenocarcinomas (24%), leukemia (12%) and other solid tumors including neuroblastoma. Characterization of T-cell lymphomas in mice overexpressing PPM1D demonstrates Pten-deletion and p53-accumulation similar to mice with p53 loss-of-function. Also, Notch1 mutations which are recurrently observed in T-cell acute lymphoblastic lymphoma (T-ALL) were frequently detected in PPM1D-transgenic mice. Hence, PPM1D acts as an oncogenic driver in connection with cellular stress, suggesting that the PPM1D gene status and expression levels should be investigated in TP53 wild-type tumors.

An unsupervised method for physical cell interaction profiling of complex tissues.

Cellular identity in complex multicellular organisms is determined in part by the physical organization of cells. However, large-scale investigation of the cellular interactome remains technically challenging. Here we develop cell interaction by multiplet sequencing (CIM-seq), an unsupervised and high-throughput method to analyze direct physical cell-cell interactions between cell types present in a tissue. CIM-seq is based on RNA sequencing of incompletely dissociated cells, followed by computational deconvolution into constituent cell types. CIM-seq estimates parameters such as number of cells and cell types in each multiplet directly from sequencing data, making it compatible with high-throughput droplet-based methods. When applied to gut epithelium or whole dissociated lung and spleen, CIM-seq correctly identifies known interactions, including those between different cell lineages and immune cells. In the colon, CIM-seq identifies a previously unrecognized goblet cell subtype expressing the wound-healing marker Plet1, which is directly adjacent to colonic stem cells. Our results demonstrate that CIM-seq is broadly applicable to unsupervised profiling of cell-type interactions in different tissue types.

Single-cell transcriptomics of human embryos identifies multiple sympathoblast lineages with potential implications for neuroblastoma origin.

Characterization of the progression of cellular states during human embryogenesis can provide insights into the origin of pediatric diseases. We examined the transcriptional states of neural crest- and mesoderm-derived lineages differentiating into adrenal glands, kidneys, endothelium and hematopoietic tissue between post-conception weeks 6 and 14 of human development. Our results reveal transitions connecting the intermediate mesoderm and progenitors of organ primordia, the hematopoietic system and endothelial subtypes. Unexpectedly, by using a combination of single-cell transcriptomics and lineage tracing, we found that intra-adrenal sympathoblasts at that stage are directly derived from nerve-associated Schwann cell precursors, similarly to local chromaffin cells, whereas the majority of extra-adrenal sympathoblasts arise from the migratory neural crest. In humans, this process persists during several weeks of development within the large intra-adrenal ganglia-like structures, which may also serve as reservoirs of originating cells in neuroblastoma.

Induction of a Timed Metabolic Collapse to Overcome Cancer Chemoresistance.

Cancer relapse begins when malignant cells pass through the extreme metabolic bottleneck of stress from chemotherapy and the byproducts of the massive cell death in the surrounding region. In acute myeloid leukemia, complete remissions are common, but few are cured. We tracked leukemia cells in vivo, defined the moment of maximal response following chemotherapy, captured persisting cells, and conducted unbiased metabolomics, revealing a metabolite profile distinct from the pre-chemo growth or post-chemo relapse phase. Persisting cells used glutamine in a distinctive manner, preferentially fueling pyrimidine and glutathione generation, but not the mitochondrial tricarboxylic acid cycle. Notably, malignant cell pyrimidine synthesis also required aspartate provided by specific bone marrow stromal cells. Blunting glutamine metabolism or pyrimidine synthesis selected against residual leukemia-initiating cells and improved survival in leukemia mouse models and patient-derived xenografts. We propose that timed cell-intrinsic or niche-focused metabolic disruption can exploit a transient vulnerability and induce metabolic collapse in cancer cells to overcome chemoresistance.

Aldehyde dehydrogenase 3a2 protects AML cells from oxidative death and the synthetic lethality of ferroptosis inducers.

Metabolic alterations in cancer represent convergent effects of oncogenic mutations. We hypothesized that a metabolism-restricted genetic screen, comparing normal primary mouse hematopoietic cells and their malignant counterparts in an ex vivo system mimicking the bone marrow microenvironment, would define distinctive vulnerabilities in acute myeloid leukemia (AML). Leukemic cells, but not their normal myeloid counterparts, depended on the aldehyde dehydrogenase 3a2 (Aldh3a2) enzyme that oxidizes long-chain aliphatic aldehydes to prevent cellular oxidative damage. Aldehydes are by-products of increased oxidative phosphorylation and nucleotide synthesis in cancer and are generated from lipid peroxides underlying the non-caspase-dependent form of cell death, ferroptosis. Leukemic cell dependence on Aldh3a2 was seen across multiple mouse and human myeloid leukemias. Aldh3a2 inhibition was synthetically lethal with glutathione peroxidase-4 (GPX4) inhibition; GPX4 inhibition is a known trigger of ferroptosis that by itself minimally affects AML cells. Inhibiting Aldh3a2 provides a therapeutic opportunity and a unique synthetic lethality to exploit the distinctive metabolic state of malignant cells.