The stress response regulator HSF1 modulates natural killer cell anti-tumour immunity.

Diverse cellular insults converge on activation of the heat shock factor 1 (HSF1), which regulates the proteotoxic stress response to maintain protein homoeostasis. HSF1 regulates numerous gene programmes beyond the proteotoxic stress response in a cell-type- and context-specific manner to promote malignancy. However, the role(s) of HSF1 in immune populations of the tumour microenvironment remain elusive. Here, we leverage an in vivo model of HSF1 activation and single-cell transcriptomic tumour profiling to show that augmented HSF1 activity in natural killer (NK) cells impairs cytotoxicity, cytokine production and subsequent anti-tumour immunity. Mechanistically, HSF1 directly binds and regulates the expression of key mediators of NK cell effector function. This work demonstrates that HSF1 regulates the immune response under the stress conditions of the tumour microenvironment. These findings have important implications for enhancing the efficacy of adoptive NK cell therapies and for designing combinatorial strategies including modulators of NK cell-mediated tumour killing.

Clonal evolution of the 3D chromatin landscape in patients with relapsed pediatric B-cell acute lymphoblastic leukemia.

Relapsed pediatric B-cell acute lymphoblastic leukemia (B-ALL) remains one of the leading causes of cancer mortality in children. We performed Hi-C, ATAC-seq, and RNA-seq on 12 matched diagnosis/relapse pediatric leukemia specimens to uncover dynamic structural variants (SVs) and 3D chromatin rewiring that may contribute to relapse. While translocations are assumed to occur early in leukemogenesis and be maintained throughout progression, we discovered novel, dynamic translocations and confirmed several fusion transcripts, suggesting functional and therapeutic relevance. Genome-wide chromatin remodeling was observed at all organizational levels: A/B compartments, TAD interactivity, and chromatin loops, including some loci shared by 25% of patients. Shared changes were found to drive the expression of genes/pathways previously implicated in resistance as well as novel therapeutic candidates, two of which (ATXN1 and MN1) we functionally validated. Overall, these results demonstrate chromatin reorganization under the selective pressure of therapy and offer the potential for discovery of novel therapeutic interventions.

circPCMTD1 : A protein-coding circular RNA that regulates DNA damage response in BCR/ABL -positive leukemias.

Circular RNAs are a novel class of RNA transcripts, which regulate important cellular functions in health and disease. Herein, we report on the functional relevance of the circPCMTD1 transcript in acute leukemias. In screening experiments, we found that circPCMTD1 depletion strongly inhibited the proliferative capacity of leukemic cells with BCR-ABL translocations. Mass cytometry experiments identified the aberrant activation of the DNA damage response as an early downstream event of circPCMTD1 depletion. In in vivo experiments, circPCMTD1 targeting prolonged the survival of mice engrafted with leukemic blasts harboring the Philadelphia chromosome. Mechanistically, we found that circPCMTD1 was enriched in the cytoplasm and associated with the ribosomes of the leukemic cells. We detected a cryptic open reading frame within the circPCMTD1 sequence and found that circPCMTD1 could generate a peptide product. The circPCMTD 1-derived peptide interacted with proteins of the BTR complex and enhanced BTR complex formation, thereby increasing tolerance to genotoxic stress.

Interferon-sensitized hematopoietic progenitors dynamically alter organismal immunity.

Inflammation has enduring impacts on organismal immunity. However, the precise mechanisms by which tissue-restricted inflammation conditions systemic responses are poorly understood. Here, we leveraged a highly compartmentalized model of skin inflammation and identified a surprising type I interferon (IFN)- mediated activation of hematopoietic stem/progenitor cells (HSPCs) that results in profound changes to systemic host responses. Post-inflamed mice were protected from atherosclerosis and had worse outcomes following influenza virus infection. This IFN-mediated HSPC modulation was dependent on IFNAR signaling and could be recapitulated with the administration of recombinant IFNα. Importantly, the transfer of post-inflamed HSPCs was sufficient to transmit the immune suppression phenotype. IFN modulation of HSPCs was rooted both in long-term changes in chromatin accessibility and the emergence of an IFN- responsive functional state from multiple progenitor populations. Collectively, our data reveal the profound and enduring effect of transient inflammation and more specifically type I IFN signaling and set the stage for a more nuanced understanding of HSPC functional modulation by peripheral immune signals.

Hematopoietic Clonal Evolution Goes Spatial.

SUMMARY: The spatial distribution of cells carrying clonal hematopoiesis mutations in the bone marrow and the potential role of interactions with the microenvironment are largely unknown. This study takes clonal evolution to the spatial level by describing a novel technique examining the spatial location of mutated clones in the bone marrow and the first evidence that mutated hematopoietic clones are spatially constrained and have heterogenous locations within millimeters of distance. See related article by Young et al., p. 153 (10).

Targeting MHC-I inhibitory pathways for cancer immunotherapy.

The MHC-I antigen presentation (AP) pathway is key to shaping mammalian CD8+ T cell immunity, with its aberrant expression closely linked to low tumor immunogenicity and immunotherapy resistance. While significant attention has been given to genetic mutations and downregulation of positive regulators that are essential for MHC-I AP, there is a growing interest in understanding how tumors actively evade MHC-I expression and/or AP through the induction of MHC-I inhibitory pathways. This emerging field of study may offer more viable therapeutic targets for future cancer immunotherapy. Here, we explore potential mechanisms by which cancer cells evade MHC-I AP and function and propose therapeutic strategies that might target these MHC-I inhibitors to restore impaired T cell immunity within the tumor microenvironment (TME).

A membrane-associated MHC-I inhibitory axis for cancer immune evasion.

Immune-checkpoint blockade has revolutionized cancer treatment, but some cancers, such as acute myeloid leukemia (AML), do not respond or develop resistance. A potential mode of resistance is immune evasion of T cell immunity involving aberrant major histocompatibility complex class I (MHC-I) antigen presentation (AP). To map such mechanisms of resistance, we identified key MHC-I regulators using specific peptide-MHC-I-guided CRISPR-Cas9 screens in AML. The top-ranked negative regulators were surface protein sushi domain containing 6 (SUSD6), transmembrane protein 127 (TMEM127), and the E3 ubiquitin ligase WWP2. SUSD6 is abundantly expressed in AML and multiple solid cancers, and its ablation enhanced MHC-I AP and reduced tumor growth in a CD8+ T cell-dependent manner. Mechanistically, SUSD6 forms a trimolecular complex with TMEM127 and MHC-I, which recruits WWP2 for MHC-I ubiquitination and lysosomal degradation. Together with the SUSD6/TMEM127/WWP2 gene signature, which negatively correlates with cancer survival, our findings define a membrane-associated MHC-I inhibitory axis as a potential therapeutic target for both leukemia and solid cancers.

A bioengineered immunocompetent human leukemia chip for preclinical screening of CAR T cell immunotherapy.

Chimeric antigen receptor (CAR) T cell immunotherapy is promising for treatment of blood cancers; however, clinical benefits remain unpredictable, necessitating development of optimal CAR T cell products. Unfortunately, current preclinical evaluation platforms are inadequate due to their limited physiological relevance to humans. We herein engineered an organotypic immunocompetent chip that recapitulates microarchitectural and pathophysiological characteristics of human leukemia bone marrow stromal and immune niches for CAR T cell therapy modeling. This leukemia chip empowered real-time spatiotemporal monitoring of CAR T cell functionality, including T cell extravasation, recognition of leukemia, immune activation, cytotoxicity, and killing. We next on-chip modelled and mapped different responses post CAR T cell therapy, i.e., remission, resistance, and relapse as observed clinically and identify factors that potentially drive therapeutic failure. Finally, we developed a matrix-based analytical and integrative index to demarcate functional performance of CAR T cells with different CAR designs and generations produced from healthy donors and patients. Together, our chip introduces an enabling '(pre-)clinical-trial-on-chip' tool for CAR T cell development, which may translate to personalized therapies and improved clinical decision-making.

Mitophagy Promotes Resistance to BH3 Mimetics in Acute Myeloid Leukemia.

BH3 mimetics are used as an efficient strategy to induce cell death in several blood malignancies, including acute myeloid leukemia (AML). Venetoclax, a potent BCL-2 antagonist, is used clinically in combination with hypomethylating agents for the treatment of AML. Moreover, MCL1 or dual BCL-2/BCL-xL antagonists are under investigation. Yet, resistance to single or combinatorial BH3-mimetic therapies eventually ensues. Integration of multiple genome-wide CRISPR/Cas9 screens revealed that loss of mitophagy modulators sensitizes AML cells to various BH3 mimetics targeting different BCL-2 family members. One such regulator is MFN2, whose protein levels positively correlate with drug resistance in patients with AML. MFN2 overexpression is sufficient to drive resistance to BH3 mimetics in AML. Insensitivity to BH3 mimetics is accompanied by enhanced mitochondria-endoplasmic reticulum interactions and augmented mitophagy flux, which acts as a prosurvival mechanism to eliminate mitochondrial damage. Genetic or pharmacologic MFN2 targeting synergizes with BH3 mimetics by impairing mitochondrial clearance and enhancing apoptosis in AML. SIGNIFICANCE: AML remains one of the most difficult-to-treat blood cancers. BH3 mimetics represent a promising therapeutic approach to eliminate AML blasts by activating the apoptotic pathway. Enhanced mitochondrial clearance drives resistance to BH3 mimetics and predicts poor prognosis. Reverting excessive mitophagy can halt BH3-mimetic resistance in AML. This article is highlighted in the In This Issue feature, p. 1501.

The Role of Inflammation in the Initiation and Progression of Myeloid Neoplasms.

Myeloid malignancies are devastating hematologic cancers with limited therapeutic options. Inflammation is emerging as a novel driver of myeloid malignancy, with important implications for tumor composition, immune response, therapeutic options, and patient survival. Here, we discuss the role of inflammation in normal and malignant hematopoiesis, from clonal hematopoiesis to full-blown myeloid leukemia. We discuss how inflammation shapes clonal output from hematopoietic stem cells, how inflammation alters the immune microenvironment in the bone marrow, and novel therapies aimed at targeting inflammation in myeloid disease. SIGNIFICANCE: Inflammation is emerging as an important factor in myeloid malignancies. Understanding the role of inflammation in myeloid transformation, and the interplay between inflammation and other drivers of leukemogenesis, may yield novel avenues for therapy.

Oncogenic drivers dictate immune control of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a genetically heterogeneous, aggressive hematological malignancy induced by distinct oncogenic driver mutations. The effect of specific AML oncogenes on immune activation or suppression is unclear. Here, we examine immune responses in genetically distinct models of AML and demonstrate that specific AML oncogenes dictate immunogenicity, the quality of immune response and immune escape through immunoediting. Specifically, expression of NrasG12D alone is sufficient to drive a potent anti-leukemia response through increased MHC Class II expression that can be overcome with increased expression of Myc. These data have important implications for the design and implementation of personalized immunotherapies for patients with AML.

Stepwise activities of mSWI/SNF family chromatin remodeling complexes direct T cell activation and exhaustion.

Highly coordinated changes in gene expression underlie T cell activation and exhaustion. However, the mechanisms by which such programs are regulated and how these may be targeted for therapeutic benefit remain poorly understood. Here, we comprehensively profile the genomic occupancy of mSWI/SNF chromatin remodeling complexes throughout acute and chronic T cell stimulation, finding that stepwise changes in localization over transcription factor binding sites direct site-specific chromatin accessibility and gene activation leading to distinct phenotypes. Notably, perturbation of mSWI/SNF complexes using genetic and clinically relevant chemical strategies enhances the persistence of T cells with attenuated exhaustion hallmarks and increased memory features in vitro and in vivo. Finally, pharmacologic mSWI/SNF inhibition improves CAR-T expansion and results in improved anti-tumor control in vivo. These findings reveal the central role of mSWI/SNF complexes in the coordination of T cell activation and exhaustion and nominate small-molecule-based strategies for the improvement of current immunotherapy protocols.

Cell-type-specific prediction of 3D chromatin organization enables high-throughput in silico genetic screening.

Investigating how chromatin organization determines cell-type-specific gene expression remains challenging. Experimental methods for measuring three-dimensional chromatin organization, such as Hi-C, are costly and have technical limitations, restricting their broad application particularly in high-throughput genetic perturbations. We present C.Origami, a multimodal deep neural network that performs de novo prediction of cell-type-specific chromatin organization using DNA sequence and two cell-type-specific genomic features-CTCF binding and chromatin accessibility. C.Origami enables in silico experiments to examine the impact of genetic changes on chromatin interactions. We further developed an in silico genetic screening approach to assess how individual DNA elements may contribute to chromatin organization and to identify putative cell-type-specific trans-acting regulators that collectively determine chromatin architecture. Applying this approach to leukemia cells and normal T cells, we demonstrate that cell-type-specific in silico genetic screening, enabled by C.Origami, can be used to systematically discover novel chromatin regulation circuits in both normal and disease-related biological systems.

An inflammatory state remodels the immune microenvironment and improves risk stratification in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a hematopoietic malignancy with poor prognosis and limited treatment options. Here we provide a comprehensive census of the bone marrow immune microenvironment in adult and pediatric patients with AML. We characterize unique inflammation signatures in a subset of AML patients, associated with inferior outcomes. We identify atypical B cells, a dysfunctional B-cell subtype enriched in patients with high-inflammation AML, as well as an increase in CD8+GZMK+ and regulatory T cells, accompanied by a reduction in T-cell clonal expansion. We derive an inflammation-associated gene score (iScore) that associates with poor survival outcomes in patients with AML. Addition of the iScore refines current risk stratifications for patients with AML and may enable identification of patients in need of more aggressive treatment. This work provides a framework for classifying patients with AML based on their immune microenvironment and a rationale for consideration of the inflammatory state in clinical settings.

A RORγt+ cell instructs gut microbiota-specific Treg cell differentiation.

The mutualistic relationship of gut-resident microbiota and the host immune system promotes homeostasis that ensures maintenance of the microbial community and of a largely non-aggressive immune cell compartment1,2. The consequences of disturbing this balance include proximal inflammatory conditions, such as Crohn's disease, and systemic illnesses. This equilibrium is achieved in part through the induction of both effector and suppressor arms of the adaptive immune system. Helicobacter species induce T regulatory (Treg) and T follicular helper (TFH) cells under homeostatic conditions, but induce inflammatory T helper 17 (TH17) cells when induced Treg (iTreg) cells are compromised3,4. How Helicobacter and other gut bacteria direct T cells to adopt distinct functions remains poorly understood. Here we investigated the cells and molecular components required for iTreg cell differentiation. We found that antigen presentation by cells expressing RORγt, rather than by classical dendritic cells, was required and sufficient for induction of Treg cells. These RORγt+ cells-probably type 3 innate lymphoid cells and/or Janus cells5-require the antigen-presentation machinery, the chemokine receptor CCR7 and the TGFß activator αv integrin. In the absence of any of these factors, there was expansion of pathogenic TH17 cells instead of iTreg cells, induced by CCR7-independent antigen-presenting cells. Thus, intestinal commensal microbes and their products target multiple antigen-presenting cells with pre-determined features suited to directing appropriate T cell differentiation programmes, rather than a common antigen-presenting cell that they endow with appropriate functions.

NUDT21 limits CD19 levels through alternative mRNA polyadenylation in B cell acute lymphoblastic leukemia.

B cell progenitor acute lymphoblastic leukemia (B-ALL) treatment has been revolutionized by T cell-based immunotherapies-including chimeric antigen receptor T cell therapy (CAR-T) and the bispecific T cell engager therapeutic, blinatumomab-targeting surface glycoprotein CD19. Unfortunately, many patients with B-ALL will fail immunotherapy due to 'antigen escape'-the loss or absence of leukemic CD19 targeted by anti-leukemic T cells. In the present study, we utilized a genome-wide CRISPR-Cas9 screening approach to identify modulators of CD19 abundance on human B-ALL blasts. These studies identified a critical role for the transcriptional activator ZNF143 in CD19 promoter activation. Conversely, the RNA-binding protein, NUDT21, limited expression of CD19 by regulating CD19 messenger RNA polyadenylation and stability. NUDT21 deletion in B-ALL cells increased the expression of CD19 and the sensitivity to CD19-specific CAR-T and blinatumomab. In human B-ALL patients treated with CAR-T and blinatumomab, upregulation of NUDT21 mRNA coincided with CD19 loss at disease relapse. Together, these studies identify new CD19 modulators in human B-ALL.

The Impact of Inflammation-Induced Tumor Plasticity during Myeloid Transformation.

Clonal hematopoiesis (CH) is an aging-associated condition characterized by the clonal outgrowth of mutated preleukemic cells. Individuals with CH are at an increased risk of developing hematopoietic malignancies. Here, we describe a novel animal model carrying a recurrent TET2 missense mutation frequently found in patients with CH and leukemia. In a fashion similar to CH, animals show signs of disease late in life when they develop a wide range of myeloid neoplasms, including acute myeloid leukemia (AML). Using single-cell transcriptomic profiling of the bone marrow, we show that disease progression in aged animals correlates with an enhanced inflammatory response and the emergence of an aberrant inflammatory monocytic cell population. The gene signature characteristic of this inflammatory population is associated with poor prognosis in patients with AML. Our study illustrates an example of collaboration between a genetic lesion found in CH and inflammation, leading to transformation and the establishment of blood neoplasms. SIGNIFICANCE: Progression from a preleukemic state to transformation, in the presence of TET2 mutations, is coupled with the emergence of inflammation and a novel population of inflammatory monocytes. Genes characteristic of this inflammatory population are associated with the worst prognosis in patients with AML. These studies connect inflammation to progression to leukemia. See related commentary by Pietras and DeGregori, p. 2234 . This article is highlighted in the In This Issue feature, p. 2221.