Myeloid malignancies are heterogenous disorders characterized by distinct molecular drivers but share convergence of oncogenic signaling pathways and propagation by ripe pro-inflammatory niches. Here, we establish a comprehensive transcriptional atlas across the spectrum of myeloproliferative neoplasms (MPN) and secondary acute myeloid leukemia (sAML) through RNA-sequencing of 158 primary samples encompassing CD34+ hematopoietic stem/progenitor cells and CD14+ monocytes. Supported by mass cytometry (CyTOF) profiling, we reveal aberrant networks of PI3K/AKT/mTOR signalling and NFκB-mediated hyper-inflammation. Combining ATAC-Seq, CUT Tag, RNA-seq, and CyTOF, we demonstrate that targeting of ribosomal protein S6 kinase A1 (RSK1) suppresses NFκB activation and diminishes pro-inflammatory mediators including tumor necrosis factor (TNF) associated with MPN disease severity and transformation. We further evaluate a therapeutic approach utilizing a first-in-class RSK inhibitor, PMD-026, currently in Phase 2 development for breast cancer, for use in myeloid malignancies. Treatment with PMD-026 suppressed disease burden across seven syngeneic and patient-derived xenograft leukemia mouse models spanning the spectrum of driver and disease-modifying mutations. These findings uncover a therapeutic avenue for a conserved dependency across MPN and sAML. Secondary acute myeloid leukemias (sAMLs) evolving from myeloproliferative neoplasms (MPNs) associate with poor prognosis. Here authors identify RSK1 as a vulnerability for MPN and sAML and show the efficacy of a first-in-class RSK inhibitor, PMD-026, against these types of myeloid malignancies.

FH, SL and BG equal contributors Myeloproliferative neoplasms (MPN) and acute myeloid leukemia (AML) exhibit shared hallmarks of hyperinflammation and aberrant clonal expansion. Chronic MPNs exhibit a propensity for transformation to secondary AML (sAML), which imparts a dismal prognosis with limited treatment options. However, the pathogenesis of leukemia transition for MPN and the bidirectional relationship between MPN and AML remain incompletely understood. In this study, we investigated commonly-featured genes and pathways of these two diseases to explore novel therapeutic strategies. To dissect the causal association between MPN and AML, we first performed two-sample bidirectional mendelian randomization (MR) analysis which revealed that MPN predicts the risk of AML across multiple statistical methods with consistent directionality and significance. Heterogeneity, pleiotropy, or outlier effects were ruled out by sensitivity analyses with no reverse causality from AML to MPN. To identify a potential myeloid signature, we estimated causal effects of 2,940 plasma proteins on MPN via a two-sample MR framework and identified 55 candidates significantly associated with increased risk of MPN (P < 0.05, OR > 1). Utilizing data from the BEAT-AML cohort, we further screened the prognostic value of these candidates using Cox and log-rank test survival analyses and identified a 4-gene myeloid signature comprising MPO, CDCP1, CRISP3 and DXCR, with each conferring prognostic significance individually. Our scRNA-seq results confirmed enrichment of myeloid signature genes in myeloid progenitor cells, with further elevation in MPN. Of note, expression of myeloid signature genes was induced by Jak2V617F and MPLW515L in Ba/F3 cells. We then investigated the combined prognostic significance of these four genes by constructing a risk score via LASSO regression modeling and divided patients into high- and low-risk groups accordingly. Cox analysis validated the risk score as an independent prognostic factor (HR: 3.01 (1.2 - 7.6), P = 0.019) and Kaplan-Meier survival analysis demonstrated that low-risk AML patients had a significantly better survival than high-risk AML patients with median survival of 21.9 months vs. 11.9 months (P < 0.0001). The prediction accuracy of the myeloid signature was successfully validated in the TCGA-LAML cohort. The risk score significantly correlated with several key clinical parameters, including age, platelet count, ELN2017 staging, monocyte percentage and bone marrow cellularity. To interrogate the underlying mechanism of the myeloid signature, we performed gene set enrichment analysis (GSEA) and observed enrichment of several inflammatory pathways in high-risk AML patients. A similar enrichment of inflammatory pathways in MPN samples compared to healthy donors observed in our previous scRNA-seq results reinforced hyperinflammation as a shared etiology of myeloid malignancies. To explore potential therapeutic strategies, we performed in silico drug sensitivity screening which predicted specific vulnerability of high-risk AML to compounds targeting PI3K/AKT/mTOR pathway signaling. Activation of PI3K/AKT/mTOR signaling in both MPN and AML samples observed across multiple single cell and bulk RNA-seq datasets further prompted us to evaluate targeted therapies against this pathway in myeloid malignancies. MPN and AML cells exhibited sensitivity to mTOR inhibitors as indicated by suppression of cellular proliferation, metabolism, colony formation, and inflammatory cytokine secretion in conjunction with induction of apoptosis. We further evaluated the therapeutic effects of mTOR inhibition in vivo via administration of omipalisib, a potent dual PI3K/mTOR inhibitor, to JAK2V617F knock-in mice. Omipalisib treatment significantly ameliorated features of myeloid malignancies including splenomegaly and leukocytosis which were both significantly reduced following 4 weeks of treatment, without affecting body weight. In summary, our MR analyses reveal that MPN predicts the risk of AML and enabled the construction of a novel myeloid signature which risk stratified AML patients across two different cohorts. We further demonstrate shared activation of PI3K/AKT/mTOR pathway across myeloid malignancies, with in vitro and in vivo data providing a rationale for therapeutic targeting of PI3K/AKT/mTOR pathway in these diseases.

JDP and STO co-corresponding authors Previous studies by our group and others have elucidated a key role for monocytes in driving hyperinflammation in myeloproliferative neoplasms (MPNs). To further interrogate the cellular landscape of aberrant inflammation in MPNs, we subjected MPN patient samples to single cell RNA-seq (scRNA-seq) analysis and found significant enrichment of inflammation-related genes, including galectins, in MPN patient monocytes. Cell-cell communication networks inferred from expression of ligands and receptors predicted monocytes as a pivotal mediator of cell interactions and galectin signaling as one of the most robust input/output pathways for monocytes. Galectins are a class of proteins that bind to glycosylated proteins and mediate broad biological functions, including cell proliferation, apoptosis, adhesion, and inflammation. Dysregulation of galectins and global protein glycosylation has been reported in various cancers, but its role in MPN has remained incompletely understood. To validate our scRNA-seq findings, we performed flow cytometry analysis which demonstrated elevated expression of galectin-1 (Gal-1) in MPN patient CD14+ monocytes. Increased plasma levels of Gal-1 were also identified in MPN patients compared to healthy individuals. We also observed enrichment of Gal-1 in mouse CD11b+ myeloid cells, with further elevation in JAK2V617F knock-in mice compared to wild-type controls. Additionally, Gal-1 expression was induced by MPLW515L and JAK2V617F and inhibited by ruxolitinib, a JAK inhibitor, in Ba/F3 cells. Thus, our results confirm enrichment of Gal-1 in MPN monocytes, with evidence of direct contribution from specific MPN driver mutations. To comprehensively characterize functional effects of Gal-1, we performed mass cytometry (CyTOF) analysis of MPN samples which demonstrated that Gal-1 stimulation induced multiple inflammatory cytokines, including TNF and IL-6, in monocytes specifically without affecting other types of cells. We further incubated CD14+ monocytes from MPN patients with recombinant Gal-1 (rGal-1) and observed markedly stimulated transcription and secretion of inflammatory cytokines, such as IL-1α, IL-1β, IL-6, IL-8 and TNF. Pharmacologic inhibition of Gal-1 by OTX008 suppressed the expression and secretion of inflammatory cytokines in MPN monocytes and monocytic cell lines. Furthermore, we observed crosstalk between LPS-TLR4 and Gal-1 signaling pathways. Using protein 3D structure prediction and co-immunoprecipitation, we confirmed interaction of Gal-1 with TLR4. Notably, targeting TLR4 via both neutralizing antibody and pharmacologic inhibition (TAK-242) abrogated the proinflammatory effects of Gal-1 on monocytes. Taken together, these findings uncover a monocyte-specific pro-inflammatory effect of Gal-1 mediated by TLR4 in MPN. To explore molecular mechanisms underlying its proinflammatory functions, we performed gene co-expression analysis for Gal-1 in monocytes and identified OXPHOS and PI3K-AKT-mTOR signaling pathways as top candidates. Both genetic and pharmacologic inhibition of Gal-1 reduced cellular ATP levels and oxygen consumption rate, suggesting a metabolic reprogramming by Gal-1 in MPN. Incubation of monocytic cell lines and MPN monocytes with rGal-1 stimulated PI3K-AKT-mTOR signaling, represented by increased levels of phosphorylated mTOR, AKT and S6. Consistently, genetic and pharmacologic inhibition of Gal-1 inhibited activation of the PI3K-AKT-mTOR pathway. Across in vivo models, both Gal-1 knockout and OTX008 ameliorated key MPN disease features, including leukocytosis and splenomegaly, driven by MPLW515L and JAK2V617F. Gal-1 inhibition also suppressed carrageenan-induced thrombosis and inflammation in mice. We further evaluated therapeutic effects of targeting global glycosylation in MPN via 2-Deoxy-D-glucose (2-DG), a global glycosylation inhibitor, which decreased splenomegaly and reduced elevated platelets and hematocrit in JAK2V617F knock-in mice. In summary, we identify enrichment of Gal-1 in MPN monocytes, potentially due to activation of JAK2-driven signaling. We further demonstrate that Gal-1 fuels monocyte inflammation by interacting with TLR4 and activation of OXPHOS and PI3K-AKT-mTOR signaling pathways. Our results uncover a novel therapeutic avenue for targeting aberrant Gal-1 and global glycosylation in MPNs.

Substantial evidence suggests a role for immunotherapy in treating Alzheimer's disease (AD). While the precise pathophysiology of AD is incompletely understood, clinical trials of antibodies targeting aggregated forms of β amyloid (Aβ) have shown that reducing amyloid plaques can mitigate cognitive decline in patients with early-stage AD. Here, we describe what we believe to be a novel approach to target and degrade amyloid plaques by genetically engineering macrophages to express an Aβ-targeting chimeric antigen receptor (CAR-Ms). When injected intrahippocampally, first-generation CAR-Ms have limited persistence and fail to significantly reduce plaque load, which led us to engineer next-generation CAR-Ms that secrete M-CSF and self-maintain without exogenous cytokines. Cytokine secreting "reinforced CAR-Ms" have greater survival in the brain niche and significantly reduce plaque load locally in vivo. These findings support CAR-Ms as a platform to rationally target, resorb, and degrade pathogenic material that accumulates with age, as exemplified by targeting Aβ in AD.

Substantial evidence suggests a role for immunotherapy in treating Alzheimer’s disease (AD). Several monoclonal antibodies targeting aggregated forms of beta amyloid (Aβ), have been shown to reduce amyloid plaques and in some cases, mitigate cognitive decline in early-stage AD patients. We sought to determine if genetically engineered macrophages could improve the targeting and degradation of amyloid plaques. Chimeric antigen receptor macrophages (CAR-Ms), which show promise as a cancer treatment, are an appealing strategy to enhance target recognition and phagocytosis of amyloid plaques in AD. We genetically engineered macrophages to express a CAR containing the anti-amyloid antibody aducanumab as the external domain and the Fc receptor signaling domain internally. CAR-Ms recognize and degrade Aβ in vitro and on APP/PS1 brain slices ex vivo; however, when injected intrahippocampally, these first-generation CAR-Ms have limited persistence and fail to reduce plaque load. We overcame this limitation by creating CAR-Ms that secrete M-CSF and self-maintain without exogenous cytokines. These CAR-Ms have greater survival in the brain niche, and significantly reduce plaque load locally in vivo . These proof-of-principle studies demonstrate that CAR-Ms, previously only applied to cancer, may be utilized to target and degrade unwanted materials, such as amyloid plaques in the brains of AD mice. Amyloid targeting CAR Macrophages engineered to secrete M-CSF promote their own local survival and expansion while resorbing amyloid plaques in the brains of Alzheimer’s disease APP/PS1 mice, resulting in significant local clearance of amyloid plaques of all sizes.

Chimeric antigen receptor (CAR) T-cell therapy represents a major advancement for hematologic malignancies, with some patients achieving long-term remission. However, the majority of treated patients still die of their disease. A consistent predictor of response is tumor quantity, wherein a higher disease burden before CAR T-cell therapy portends a worse prognosis. Focal radiation to bulky sites of the disease can decrease tumor quantity before CAR T-cell therapy, but whether this strategy improves survival is unknown. We find that substantially reducing systemic tumor quantity using high-dose radiation to areas of bulky disease, which is commonly done clinically, is less impactful on overall survival in mice achieved by CAR T cells than targeting all sites of disease with low-dose total tumor irradiation (TTI) before CAR T-cell therapy. This finding highlights another predictor of response, tumor quality, the intrinsic resistance of an individual patient's tumor cells to CAR T-cell killing. Little is known about whether or how an individual tumor's intrinsic resistance may change under different circumstances. We find a transcriptional "death receptor score" that reflects a tumor's intrinsic sensitivity to CART cells can be temporarily increased by low-dose TTI, and the timing of this transcriptional change correlates with improved in vivo leukemia control by an otherwise limited number of CAR T cells. This suggests an actionable method for potentially improving outcomes in patients predicted to respond poorly to this promising therapy and highlights that intrinsic tumor attributes may be equally or more important predictors of CAR T-cell response as tumor burden.

Abstract Background T helper 17 (Th17) cells play an important role in barrier protection in the gastrointestinal tract but are also key pathological drivers of Inflammatory Bowel Disease (IBD). Although a number of transcription factors governing Th17 differentiation have been identified, the intracellular signalling pathways regulating Th17 differentiation are poorly understood. Hedgehog (Hh) signalling controls cell-fate choices in numerous tissue compartments and is targetable by highly selective, clinically-approved small molecule inhibitors. However the role of Hh signalling in Th17 differentiation and effector function is unstudied. Methods We generated two conditional knockout mouse models targeting Hh signalling components Smo and Ihh to study Th17 differentiation in vitro by flow cytometry and gene expression analysis. For in vivo studies, T cell adoptive transfer colitis was performed using donor Ihh knockout T cells or heterozygote controls. Histological analysis, mouse weight, colon length/weight measurements, and flow cytometric analysis was performed. We supplement this with the use of two small-molecule Smo antagonists for in vitro and in vivo studies of Th17 function. To underscore the translational relevance of our findings, we conducted bioinformatic analyses of published gene expression datasets of human rectal biopsies from two large independent cohorts of Ulcerative Colitis patients and healthy controls. Results We find that intracellular Hh signalling, independently of extracellular Hh ligands, selectively drives differentiation and effector function of Th17 cells but not of other T helper cell lineages. We demonstrate in vivo that inhibition of the Hh pathway with either the clinically-approved small molecule inhibitor vismodegib or genetic ablation of Ihh in CD4+ T cells results in a significant decrease in histological and clinical readouts of disease severity as well as a significant reduction in IL-17a+ Th17 cells. Our bioinformatic analyses show that Hh component expression levels are upregulated in human Ulcerative Colitis patient samples and are closely correlated with expression of Th17 markers. Mechanistically we show that the T-cell-intrinsic Indian Hedgehog (Ihh) ligand signals via the signal transducer Smoothened to activate both canonical and non-canonical Hh pathways, through the Gli3 transcription factor and AMPK phosphorylation, respectively. Conclusion We uncover Hh signalling as a novel pathway controlling Th17 differentiation and pathogenicity in IBD with Gli3 acting as a newly-identified crucial regulatory transcription factor. Our work paves the way for the use of Hh inhibitors for the treatment of IBD.

Abstract BACKGROUND T helper 17 (Th17) cells play a key role in barrier protection in the gastrointestinal tract but are also key pathological drivers of Inflammatory Bowel Disease (IBD). Although a number of key transcription factors governing Th17 differentiation have been identified, the intracellular signaling pathways regulating Th17 differentiation are poorly understood. Given the highly druggable nature of many intracellular signaling pathways, understanding the signaling pathways involved in Th17 differentiation holds great promise to identify novel drug targets for the treatment of IBD. Hedgehog (Hh) signaling controls cell-fate choices in numerous tissue compartments and is targetable by highly selective, clinically-approved small molecule inhibitors. However the role of Hh signaling in Th17 differentiation and effector function is unstudied. METHODS We generated two conditional knockout mouse models targeting Hh signaling components Smo and Ihh to study Th17 differentiation and effector function in vitro and in vivo in murine T cell adoptive transfer colitis. We supplement this with the use of two small-molecule Smo antagonists for both in vitro and in vivo studies of Th17 function. To underscore the translational relevance of our findings, we have conducted bioinformatic analyses of published gene expression datasets of human rectal biopsies from two large independent cohorts of Ulcerative Colitis patients and healthy controls as well as from sorted human effector T cells from blood and lamina propria of healthy individuals. RESULTS We find that intracellular Hh signaling, independently of extracellular Hh ligands, selectively drives differentiation and effector function of Th17 cells but not of other T helper cell lineages. Using two models of intestinal inflammation, we demonstrate that inhibition of the Hh pathway with either the clinically-approved small molecule inhibitor vismodegib or genetic ablation of Ihh in CD4+ T cells profoundly diminishes disease severity and Th17-induced pathology in the intestine. Our bioinformatic analyses show that Hh component expression levels are upregulated in human ulcerative colitis patient samples and are closely correlated with expression of Th17 markers/cytokines. Mechanistically we show that the T-cell-intrinsic Indian Hedgehog (Ihh) ligand signals via the signal transducer Smoothened to activate both canonical and non-canonical Hh pathways, through the Gli3 transcription factor and AMPK phosphorylation, respectively. CONCLUSIONS We uncover Hh signaling as a novel pathway controlling Th17 differentiation and pathogenicity in IBD with Gli3 acting as a newly-identified crucial regulatory transcription factor. Our work paves the way for the use of Hh inhibitors for the treatment of IBD.

Th17 cells are key drivers of autoimmune disease. However, the signaling pathways regulating Th17 polarization are poorly understood. Hedgehog signaling regulates cell fate decisions during embryogenesis and adult tissue patterning. Here we find that cell-autonomous Hedgehog signaling, independent of exogenous ligands, selectively drives the polarization of Th17 cells but not other T helper cell subsets. We show that endogenous Hedgehog ligand, Ihh, signals to activate both canonical and non-canonical Hedgehog pathways through Gli3 and AMPK. We demonstrate that Hedgehog pathway inhibition with either the clinically-approved small molecule inhibitor vismodegib or genetic ablation of Ihh in CD4 + T cells greatly diminishes disease severity in two mouse models of intestinal inflammation. We confirm that Hedgehog pathway expression is upregulated in tissue from human ulcerative colitis patients and correlates with Th17 marker expression. This work implicates Hedgehog signaling in Th17 polarization and intestinal immunopathology and indicates the potential therapeutic use of Hedgehog inhibitors in the treatment of inflammatory bowel disease.

Abstract T helper 17 (Th17) cells play a key role in barrier protection against fungal and bacterial pathogens but are also pathological drivers of many inflammatory diseases. Although the transcription factor networks governing Th17 differentiation are well defined, the signaling pathways that regulate the development and function of this important CD4+ T cell subset are still poorly understood. Hedgehog (Hh) signaling plays important roles in regulating cell fate decisions during embryogenesis and adult tissue patterning. Using novel CD4-specific Hh knockout mice, we find that intracellular Hh signaling, independently of exogenous Hh ligands, selectively drives Th17 lineage differentiation but not the development of Th1, Th2, or iTreg CD4+ Th cells. We show that the endogenous Indian Hh (Ihh) ligand signals via the signal transducer Smoothened to activate both canonical and non-canonical Hh pathways, through the Gli3 transcription factor and AMPK phosphorylation, respectively. Using two models of intestinal inflammation, we demonstrate that inhibition of the Hh pathway with either the clinically approved small molecule inhibitor vismodegib or genetic ablation of Ihh in CD4+ T cells greatly diminishes disease severity. Taken together, we have uncovered Hh as a novel signaling pathway controlling Th17 differentiation and Gli3 as a crucial transcription factor in this process. Our work paves the way for a potential use of Hh inhibitors in the treatment of inflammatory bowel disease and other autoimmune diseases.

NKG2D is an important immunoreceptor expressed on the surface of NK cells and some T cells. NKG2D recognizes a set of ligands typically expressed on infected or transformed cells, but recent studies have also documented NKG2D ligands on subsets of host non-tumor cells in tumor-bearing animals and humans. Here we show that in transplanted tumors and genetically engineered mouse cancer models, tumor-associated macrophages are induced to express the NKG2D ligand RAE-1δ. We find that a soluble factor produced by tumor cells is responsible for macrophage RAE-1δ induction, and we identify tumor-derived colony-stimulating factor-1 (CSF-1) as necessary and sufficient for macrophage RAE-1δ induction in vitro and in vivo. Furthermore, we show that induction of RAE-1δ on macrophages by CSF-1 requires PI3K p110α kinase signaling. Thus, production of CSF-1 by tumor cells leading to activation of PI3K p110α represents a novel cellular and molecular pathway mediating NKG2D ligand expression on tumor-associated macrophages.

Natural Killer (NK) cells confer protection from tumors and infections by releasing cytotoxic granules and pro-inflammatory cytokines upon recognition of diseased cells. The responsiveness of NK cells to acute stimulation is dynamically tuned by steady-state receptor-ligand interactions of an NK cell with its cellular environment. Here, we demonstrate that in healthy WT mice the NK activating receptor NKG2D is engaged in vivo by one of its ligands, RAE-1ε, which is expressed constitutively by lymph node endothelial cells and highly induced on tumor-associated endothelium. This interaction causes internalization of NKG2D from the NK cell surface and transmits an NK-intrinsic signal that desensitizes NK cell responses globally to acute stimulation, resulting in impaired NK antitumor responses in vivo.