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Cutaneous SCC (cSCC) is the most frequently occuring skin cancer with metastatic potential and can manifest rapidly as a common side effect in patients receiving systemic kinase inhibitors. Here, we use massively parallel exome and targeted level sequencin

Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults. Long-term survival of patients with AML has changed little over the past decade, necessitating the identification and validation of new AML targets. Integration of genomic approaches with small-molecule and genetically based high-throughput screening holds the promise of improved discovery of candidate targets for cancer therapy. Here, we identified a role for glycogen synthase kinase 3α (GSK-3α) in AML by performing 2 independent small-molecule library screens and an shRNA screen for perturbations that induced a differentiation expression signature in AML cells. GSK-3 is a serine-threonine kinase involved in diverse cellular processes, including differentiation, signal transduction, cell cycle regulation, and proliferation. We demonstrated that specific loss of GSK-3α induced differentiation in AML by multiple measurements, including induction of gene expression signatures, morphological changes, and cell surface markers consistent with myeloid maturation. GSK-3α-specific suppression also led to impaired growth and proliferation in vitro, induction of apoptosis, loss of colony formation in methylcellulose, and anti-AML activity in vivo. Although the role of GSK-3β has been well studied in cancer development, these studies support a role for GSK-3α in AML. [ABSTRACT FROM AUTHOR]

Notch proteins undergo ligand-induced proteolysis to release a nuclear effector that influences a wide range of cellular processes by regulating transcription. Despite years of study, however, how Notch induces the transcription of its target genes remains unclear. Here, we comprehensively examine the response to human Notch1 across a time course of activation using high-resolution genomic assays of chromatin accessibility and nascent RNA production. Our data reveal that Notch induces target gene transcription primarily by releasing paused RNA polymerase II (RNAPII). Moreover, in contrast to prevailing models suggesting that Notch acts by promoting chromatin accessibility, we found that open chromatin was established at Notch-responsive regulatory elements prior to Notch signal induction through SWI/SNF-mediated remodeling. Together, these studies show that the nuclear response to Notch signaling is dictated by the pre-existing chromatin state and RNAPII distribution at the time of signal activation., (© 2024 Rogers et al.; Published by Cold Spring Harbor Laboratory Press.)

Mind bomb 1 (MIB1) is a RING E3 ligase that ubiquitinates Notch ligands, a necessary step for induction of Notch signaling. The structural basis for binding of the JAG1 ligand by the N-terminal region of MIB1 is known, yet how the ankyrin (ANK) and RING domains of MIB1 cooperate to catalyze ubiquitin transfer from E2∼Ub to Notch ligands remains unclear. Here, we show that the third RING domain and adjacent coiled coil region (ccRING3) drive MIB1 dimerization and that MIB1 ubiquitin transfer activity relies solely on ccRING3. We report X-ray crystal structures of a UbcH5B-ccRING3 complex and the ANK domain. Directly tethering the MIB1 N-terminal region to ccRING3 forms a minimal MIB1 protein sufficient to induce a Notch response in receiver cells and rescue mib knockout phenotypes in flies. Together, these studies define the functional elements of an E3 ligase needed for ligands to induce a Notch signaling response., Competing Interests: Declaration of interests S.C.B. is on the board of directors of the non-profit Institute for Protein Innovation and the Revson Foundation, is on the scientific advisory board for and receives funding from Erasca, Inc. for an unrelated project, is an advisor to MPM Capital, and is a consultant for IFM, Scorpion Therapeutics, Odyssey Therapeutics, Droia Ventures, and Ayala Pharmaceuticals for unrelated projects. J.C.A. is a consultant for Ayala Pharmaceuticals, Cellestia, Inc., SpringWorks Therapeutics, and Remix Therapeutics. P.A.C. has been a consultant for Scorpion Therapeutics, Nested Therapeutics, and Intonation Research Labs., (Copyright © 2024. Published by Elsevier Inc.)

PP2A serine/threonine phosphatases are heterotrimeric complexes that execute many essential physiologic functions. These activities are modulated by additional regulatory proteins, such as ARPP19, FAM122A, and IER5. Here, we report the cryoelectron microscopy structure of a complex of PP2A/B55α with the N-terminal structured region of IER5 (IER5-N50), which occludes a surface on B55α used for substrate recruitment, and show that IER5-N50 inhibits PP2A/B55α catalyzed dephosphorylation of pTau in biochemical assays. Mutations of full-length IER5 that disrupt its PP2A/B55α interface interfere with co-immunoprecipitation of PP2A/B55α. These mutations and deletions that remove the nuclear localization sequence of IER5 suppress cellular events such as KRT1 expression that depend on association of IER5 with PP2A/B55α. Querying the Alphafold2 predicted structure database identified SERTA domain proteins as high-confidence PP2A/B55α-binding structural homologs of IER5-N50. These studies define the molecular basis of PP2A/B55α inhibition by IER5-family proteins and suggest a roadmap for selective pharmacologic modulation of PP2A/B55α complexes., Competing Interests: SCB is on the board of directors of the non-profit Institute for Protein Innovation and the Revson Foundation, is on the scientific advisory board for and receives funding from Erasca, Inc. for an unrelated project, is an advisor to MPM Capital, and is a consultant for IFM, Scorpion Therapeutics, Odyssey Therapeutics, Droia Ventures, and Ayala Pharmaceuticals for unrelated projects. JCA is a consultant for Ayala Pharmaceuticals, Cellestia, Inc., SpringWorks Therapeutics, and Remix Therapeutics. The other authors declare that they have no competing interests.

Mammalian Notch signaling occurs when the binding of Delta or Jagged to Notch stimulates the proteolytic release of the Notch intracellular domain (NICD), which enters the nucleus to control target gene expression. To determine the temporal dynamics of events associated with Notch signaling under native conditions, we fluorescently tagged Notch and Delta at their endogenous genomic loci and visualized them upon pairing of receiver (Notch) and sender (Delta) cells as a function of time after cell contact. At contact sites, Notch and Delta immediately accumulated at 1:1 stoichiometry in synapses, which resolved by 15-20 min after contact. Synapse formation preceded the entrance of the Notch extracellular domain into the sender cell and accumulation of NICD in the nucleus of the receiver cell, which approached a maximum after ∼45 min and was prevented by chemical and genetic inhibitors of signaling. These findings directly link Notch-Delta synapse dynamics to NICD production with spatiotemporal precision., Competing Interests: Declaration of interests S.C.B. is on the board for the Institute for Protein Innovation and the Revson Foundation, on the SAB for MPM Capital and Erasca, and consults for Scorpion Therapeutics and Odyssey Therapeutics- on unrelated projects. T.K. is a member of the Medical Advisory Board of AI Therapeutics. J.C.A. is a consultant for Ayala Pharmaceuticals, Cellestia, SpringWorks, and Remix Therapeutics., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Purpose: This study investigated the effectiveness of algorithmic testing in hematopathology at the Brigham and Women's Hospital and Dana-Farber Cancer Institute (DFCI). The algorithm was predicated on test selection after an initial pathologic evaluation to maximize cost-effective testing, especially for expensive molecular and cytogenetic assays., Materials and Methods: Standard ordering protocols (SOPs) for 17 disease categories were developed and encoded in a decision support application. Six months of retrospective data from application beta testing was obtained and compared with actual testing practices during that timeframe. In addition, 2 years of prospective data were also obtained from patients at one community satellite site., Results: A total of 460 retrospective cases (before introduction of algorithmic testing) and 109 prospective cases (following introduction) were analyzed. In the retrospective data, 61.7% of tests (509 of 825) were concordant with the SOPs while 38.3% (316 of 825) were overordered and 30.8% (227 of 736) of SOP-recommended tests were omitted. In the prospective data, 98.8% of testing was concordant (244 of 247 total tests) with only 1.2% overordered tests (3 of 247) and 7.6% omitted tests (20 of 264 SOP-recommended tests; overall P < .001). The cost of overordered tests before implementing SOP indicates a potential annualized saving of $1,347,520 in US dollars (USD) in overordered testing at Brigham and Women's Hospital/DFCI. Only two of 316 overordered tests (0.6%) returned any additional information, both for extremely rare clinical circumstances., Conclusion: Implementation of SOPs dramatically improved test ordering practices, with a just right number of ancillary tests that minimizes cost and has no significant impact on acquiring key informative test results.

Nonhematopoietic lymph node stromal cells (LNSCs) regulate lymphocyte trafficking, survival, and function for key roles in host defense, autoimmunity, alloimmunity, and lymphoproliferative disorders. However, the study of LNSCs in human diseases is complicated by a dependence on viable lymphoid tissues, which are most often excised prior to establishment of a specific diagnosis. Here, we demonstrate that cryopreservation can be used to bank lymphoid tissue for the study of LNSCs in human disease. Using human tonsils and lymph nodes (LN), lymphoid tissue fragments were cryopreserved for subsequent enzymatic digestion and recovery of viable nonhematopoietic cells. Flow cytometry and single-cell transcriptomics identified comparable proportions of LN stromal cell types in fresh and cryopreserved tissue. Moreover, cryopreservation had little effect on transcriptional profiles, which showed significant overlap between tonsils and LN. The presence and spatial distribution of transcriptionally defined cell types were confirmed by in situ analyses. Our broadly applicable approach promises to greatly enable research into the roles of LNSCs in human disease., (© 2023 Wiley-VCH GmbH.)

Notch signaling relies on ligand-induced proteolysis of the transmembrane receptor Notch to liberate a nuclear effector that drives cell fate decisions. Upon ligand binding, sequential cleavage of Notch by the transmembrane protease ADAM10 and the intracellular protease γ-secretase releases the Notch intracellular domain (NICD), which translocates to the nucleus and forms a complex that induces target gene transcription. To map the location and timing of the individual steps required for the proteolysis and movement of Notch from the plasma membrane to the nucleus, we used proximity labeling with quantitative, multiplexed mass spectrometry to monitor the interaction partners of endogenous NOTCH2 after ligand stimulation in the presence of a γ-secretase inhibitor and as a function of time after inhibitor removal. Our studies showed that γ-secretase-mediated cleavage of NOTCH2 occurred in an intracellular compartment and that formation of nuclear complexes and recruitment of chromatin-modifying enzymes occurred within 45 min of inhibitor washout. These findings provide a detailed spatiotemporal map tracking the path of Notch from the plasma membrane to the nucleus and identify signaling events that are potential targets for modulating Notch activity.

Atrial fibrillation disrupts contraction of the atria, leading to stroke and heart failure. We deciphered how immune and stromal cells contribute to atrial fibrillation. Single-cell transcriptomes from human atria documented inflammatory monocyte and SPP1 + macrophage expansion in atrial fibrillation. Combining hypertension, obesity, and mitral valve regurgitation (HOMER) in mice elicited enlarged, fibrosed, and fibrillation-prone atria. Single-cell transcriptomes from HOMER mouse atria recapitulated cell composition and transcriptome changes observed in patients. Inhibiting monocyte migration reduced arrhythmia in Ccr2 -∕- HOMER mice. Cell-cell interaction analysis identified SPP1 as a pleiotropic signal that promotes atrial fibrillation through cross-talk with local immune and stromal cells. Deleting Spp1 reduced atrial fibrillation in HOMER mice. These results identify SPP1 + macrophages as targets for immunotherapy in atrial fibrillation.

Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) show potent efficacy in several ALK-driven tumors, but the development of resistance limits their long-term clinical impact. Although resistance mechanisms have been studied extensively in ALK-driven non-small cell lung cancer, they are poorly understood in ALK-driven anaplastic large cell lymphoma (ALCL). Here, we identify a survival pathway supported by the tumor microenvironment that activates phosphatidylinositol 3-kinase γ (PI3K-γ) signaling through the C-C motif chemokine receptor 7 (CCR7). We found increased PI3K signaling in patients and ALCL cell lines resistant to ALK TKIs. PI3Kγ expression was predictive of a lack of response to ALK TKI in patients with ALCL. Expression of CCR7, PI3Kγ, and PI3Kδ were up-regulated during ALK or STAT3 inhibition or degradation and a constitutively active PI3Kγ isoform cooperated with oncogenic ALK to accelerate lymphomagenesis in mice. In a three-dimensional microfluidic chip, endothelial cells that produce the CCR7 ligands CCL19/CCL21 protected ALCL cells from apoptosis induced by crizotinib. The PI3Kγ/δ inhibitor duvelisib potentiated crizotinib activity against ALCL lines and patient-derived xenografts. Furthermore, genetic deletion of CCR7 blocked the central nervous system dissemination and perivascular growth of ALCL in mice treated with crizotinib. Thus, blockade of PI3Kγ or CCR7 signaling together with ALK TKI treatment reduces primary resistance and the survival of persister lymphoma cells in ALCL.

Programming T cells to distinguish self from non-self is a vital, multi-step process that occurs in the thymus 1-4 . Signalling through the pre-T cell receptor (preTCR), a CD3-associated heterodimer comprising an invariant pTα chain and a clone-specific β chain, is a critical early checkpoint in thymocyte development within the αβ T cell lineage 5,6 . PreTCRs arrayed on CD4 - CD8 - double-negative thymocytes ligate peptides bound to major histocompatibility complex molecules (pMHC) on thymic stroma, similar to αβ T cell receptors that appear on CD4 + CD8 + double-positive thymocytes, but via a different molecular docking strategy 7-10 . Here we show the consequences of these distinct interactions for thymocyte progression using synchronized fetal thymic progenitor cultures that differ in the presence or absence of pMHC on support stroma, and single-cell transcriptomes at key thymocyte developmental transitions. Although major histocompatibility complex (MHC)-negative stroma fosters αβ T cell differentiation, the absence of preTCR-pMHC interactions leads to deviant thymocyte transcriptional programming associated with dedifferentiation. Highly proliferative double-negative and double-positive thymocyte subsets emerge, with antecedent characteristics of T cell lymphoblastic and myeloid malignancies. Compensatory upregulation of diverse MHC class Ib proteins in B2m/H2-Ab1 MHC-knockout mice partially safeguards in vivo thymocyte progression, although disseminated double-positive thymic tumours may develop with ageing. Thus, as well as promoting β chain repertoire broadening for subsequent αβ T cell receptor utilization, preTCR-pMHC interactions limit cellular plasticity to facilitate normal thymocyte differentiation and proliferation that, if absent, introduce developmental vulnerabilities., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Salivary adenoid cystic carcinoma (ACC) is a rare, biologically unique biphasic tumor that consists of malignant myoepithelial and luminal cells. MYB and Notch signaling have been implicated in ACC pathophysiology, but in vivo descriptions of these two programs in human tumors and investigation into their active coordination remain incomplete. We utilize single-cell RNA sequencing to profile human head and neck ACC, including a comparison of primary ACC with a matched local recurrence. We define expression heterogeneity in these rare tumors, uncovering diversity in myoepithelial and luminal cell expression. We find differential expression of Notch ligands DLL1, JAG1, and JAG2 in myoepithelial cells, suggesting a paracrine interaction that may support oncogenic Notch signaling. We validate this selective expression in three published cohorts of patients with ACC. Our data provide a potential explanation for the biphasic nature of low- and intermediate-grade ACC and may help direct new therapeutic strategies against these tumors., Competing Interests: Declaration of interests J.C.A. is a consultant to Ayala Pharmaceuticals, Remix Therapeutics, and Cellestia, Inc. B.E.B. declares outside interests in Fulcrum Therapeutics, Arsenal Biosciences, HiFiBio, Cell Signaling Technologies, and Chroma Medicine., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Loss of the gene SMARCB1 drives the development of malignant rhabdoid tumors, epithelioid sarcomas, and other malignancies. The SMARCB1 protein is a core component of the SWI/SNF (SWItch/Sucrose Non-Fermentable) family of chromatin remodeling complexes, which are important regulators of gene expression and cell differentiation. Here, we use CRISPR-Cas9 to create germline smarcb1 loss of function in zebrafish. We demonstrate that the combination of smarcb1 deficiency with mutant p53 results in the development of epithelioid sarcomas, angiosarcomas, and carcinomas of the thyroid and colon. Although human epithelioid sarcomas do not frequently harbor p53 mutations, smarcb1-deficient tumors in zebrafish were only observed following disruption of p53, indicating that p53 signaling in human tumors might be attenuated through alternative mechanisms, such as MDM2-mediated proteasomal degradation of p53. To leverage this possibility for the treatment of human epithelioid sarcoma, we tested small molecule-mediated disruption of the p53-MDM2 interaction, which stabilized p53 protein leading to p53-pathway reactivation, cell-cycle arrest, and increased apoptosis. Moreover, we found that MDM2 inhibition and the topoisomerase II inhibitor doxorubicin synergize in targeting epithelioid sarcoma cell viability. This could be especially relevant for patients with epithelioid sarcoma because doxorubicin represents the current gold standard for their clinical treatment. Our results therefore warrant reactivating p53 protein in SMARCB1-deficient, p53-wildtype epithelioid sarcomas using combined doxorubicin and MDM2 inhibitor therapy., (©2022 American Association for Cancer Research.)

Notch receptors participate in a conserved pathway in which ligands expressed on neighboring cells trigger a series of proteolytic cleavages that allow the intracellular portion of the receptor to travel to the nucleus and form a short-lived transcription complex that turns on target gene expression. The directness and seeming simplicity of this signaling mechanism belies the complexity of the outcomes of Notch signaling in normal cells, which are highly context and dosage dependent. This complexity is reflected in the diverse roles of Notch in cancers of various types, in which Notch may be oncogenic or tumor suppressive and may have a wide spectrum of effects on tumor cells and stromal elements. This review provides an overview of the roles of Notch in cancer and discusses challenges to clinical translation of Notch targeting agents as well as approaches that may overcome these hurdles., Competing Interests: Declaration of Competing Interest Jon C. Aster is a consultant for Ayala Pharmaceuticals and Cellestia, Inc., companies that are developing Notch pathway inhibitors for clinical use. He does not hold an equity position in either company. Antonio Ferreira declares that he has no conflicts of interest., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Adenoid cystic carcinoma (ACC) is an aggressive salivary gland malignancy with limited treatment options for recurrent or metastatic disease. Due to chemotherapy resistance and lack of targeted therapeutic approaches, current treatment options for the localized disease are limited to surgery and radiation, which fails to prevent locoregional recurrences and distant metastases in over 50% of patients. Approximately 20% of patients with ACC carry NOTCH-activating mutations that are associated with a distinct phenotype, aggressive disease, and poor prognosis. Given the role of NOTCH signaling in regulating tumor cell behavior, NOTCH inhibitors represent an attractive potential therapeutic strategy for this subset of ACC. AL101 (osugacestat) is a potent γ-secretase inhibitor that prevents activation of all four NOTCH receptors. While this investigational new drug has demonstrated antineoplastic activity in several preclinical cancer models and in patients with advanced solid malignancies, we are the first to study the therapeutic benefit of AL101 in ACC. Here, we describe the antitumor activity of AL101 using ACC cell lines, organoids, and patient-derived xenograft models. Specifically, we find that AL101 has potent antitumor effects in in vitro and in vivo models of ACC with activating NOTCH1 mutations and constitutively upregulated NOTCH signaling pathway, providing a strong rationale for evaluation of AL101 in clinical trials for patients with NOTCH-driven relapsed/refractory ACC., (© 2022. The Author(s).)

Diffuse large B-cell lymphoma (DLBCL) is the most common B-cell malignancy, with varying prognosis after the gold standard rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Several prognostic models have been established by focusing primarily on characteristics of lymphoma cells themselves, including cell-of-origin (COO), genomic alterations, and gene/protein expressions. However, the prognostic impact of the lymphoma microenvironment and its association with characteristics of lymphoma cells are not fully understood. Using the nCounter-based gene expression profiling of untreated DLBCL tissues, we assess the clinical impact of lymphoma microenvironment on the clinical outcomes and pathophysiological, molecular signatures in DLBCL. The presence of normal germinal center (GC)-microenvironmental cells, including follicular T cells, macrophage/dendritic cells, and stromal cells in lymphoma tissue indicates a positive therapeutic response. Our prognostic model, based on quantitation of transcripts from distinct GC-microenvironmental cell markers, clearly identified patients with graded prognosis independently of existing prognostic models. We observed increased incidences of genomic alterations and aberrant gene expression associated with poor prognosis in DLBCL tissues lacking GC-microenvironmental cells relative to those containing these cells. These data suggest that the loss of GC-associated microenvironmental signature dictates clinical outcomes of DLBCL patients reflecting the accumulation of "unfavorable" molecular signatures., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Chronic lymphocytic leukemia (CLL) is characterized by disordered DNA methylation, suggesting these epigenetic changes might play a critical role in disease onset and progression. The methyltransferase DNMT3A is a key regulator of DNA methylation. Although DNMT3A somatic mutations in CLL are rare, we found that low DNMT3A expression is associated with more aggressive disease. A conditional knockout mouse model showed that homozygous depletion of Dnmt3a from B cells results in the development of CLL with 100% penetrance at a median age of onset of 5.3 months, and heterozygous Dnmt3a depletion yields a disease penetrance of 89% with a median onset at 18.5 months, confirming its role as a haploinsufficient tumor suppressor. B1a cells were confirmed as the cell of origin of disease in this model, and Dnmt3a depletion resulted in focal hypomethylation and activation of Notch and Myc signaling. Amplification of chromosome 15 containing the Myc gene was detected in all CLL mice tested, and infiltration of high- Myc -expressing CLL cells in the spleen was observed. Notably, hyperactivation of Notch and Myc signaling was exclusively observed in the Dnmt3a CLL mice, but not in three other CLL mouse models tested ( Sf3b1-Atm , Ikzf3 , and MDR ), and Dnmt3a -depleted CLL were sensitive to pharmacologic inhibition of Notch signaling in vitro and in vivo . Consistent with these findings, human CLL samples with lower DNMT3A expression were more sensitive to Notch inhibition than those with higher DNMT3A expression. Altogether, these results suggest that Dnmt3a depletion induces CLL that is highly dependent on activation of Notch and Myc signaling. SIGNIFICANCE: Loss of DNMT3A expression is a driving event in CLL and is associated with aggressive disease, activation of Notch and Myc signaling, and enhanced sensitivity to Notch inhibition., (©2021 American Association for Cancer Research.)

Classical dendritic cells (cDC) are professional antigen-presenting cells (APC) that regulate immunity and tolerance. Neutrophil-derived cells with properties of DCs (nAPC) are observed in human diseases and after culture of neutrophils with cytokines. Here we show that FcγR-mediated endocytosis of antibody-antigen complexes or an anti-FcγRIIIB-antigen conjugate converts neutrophils into nAPCs that, in contrast to those generated with cytokines alone, activate T cells to levels observed with cDCs and elicit CD8 + T cell-dependent anti-tumor immunity in mice. Single cell transcript analyses and validation studies implicate the transcription factor PU.1 in neutrophil to nAPC conversion. In humans, blood nAPC frequency in lupus patients correlates with disease. Moreover, anti-FcγRIIIB-antigen conjugate treatment induces nAPCs that can activate autologous T cells when using neutrophils from individuals with myeloid neoplasms that harbor neoantigens or those vaccinated against bacterial toxins. Thus, anti-FcγRIIIB-antigen conjugate-induced conversion of neutrophils to immunogenic nAPCs may represent a possible immunotherapy for cancer and infectious diseases., (© 2021. The Author(s).)

Lineage plasticity and stemness have been invoked as causes of therapy resistance in cancer, because these flexible states allow cancer cells to dedifferentiate and alter their dependencies. We investigated such resistance mechanisms in relapsed/refractory early T-cell progenitor acute lymphoblastic leukemia (ETP-ALL) carrying activating NOTCH1 mutations via full-length single-cell RNA sequencing (scRNA-seq) of malignant and microenvironmental cells. We identified 2 highly distinct stem-like states that critically differed with regard to cell cycle and oncogenic signaling. Fast-cycling stem-like leukemia cells demonstrated Notch activation and were effectively eliminated in patients by Notch inhibition, whereas slow-cycling stem-like cells were Notch independent and rather relied on PI3K signaling, likely explaining the poor efficacy of Notch inhibition in this disease. Remarkably, we found that both stem-like states could differentiate into a more mature leukemia state with prominent immunomodulatory functions, including high expression of the LGALS9 checkpoint molecule. These cells promoted an immunosuppressive leukemia ecosystem with clonal accumulation of dysfunctional CD8+ T cells that expressed HAVCR2, the cognate receptor for LGALS9. Our study identified complex interactions between signaling programs, cellular plasticity, and immune programs that characterize ETP-ALL, illustrating the multidimensionality of tumor heterogeneity. In this scenario, combination therapies targeting diverse oncogenic states and the immune ecosystem seem most promising to successfully eliminate tumor cells that escape treatment through coexisting transcriptional programs., (© 2021 by The American Society of Hematology.)

Purpose: Salivary gland adenoid cystic carcinoma (ACC) has heterogeneous clinical behavior. Currently, all patients are treated uniformly, and no standard-of-care systemic therapy exists for metastatic ACC. We conducted an integrated proteogenomic analyses of ACC tumors to identify dysregulated pathways and propose a classification with therapeutic implications., Experimental Design: RNA/DNA sequencing of 54 flash-frozen salivary ACCs and reverse phase protein array (RPPA) in 38 specimens were performed, with validation by Western blotting and/or IHC. Three independent ACC cohorts were used for validation., Results: Both unbiased RNA sequencing (RNA-seq) and RPPA analysis revealed two molecular subtypes: ACC-I (37%) and ACC-II (63%). ACC-I had strong upregulation of MYC , MYC target genes, and mRNA splicing, enrichment of NOTCH -activating mutations, and dramatically worse prognosis. ACC-II exhibited upregulation of TP63 and receptor tyrosine kinases (AXL, MET, and EGFR) and less aggressive clinical course. TP63 and MYC were sufficient to assign tumors to ACC subtypes, which was validated in one independent cohort by IHC and two additional independent cohorts by RNA-seq. Furthermore, IHC staining for MYC and P63 protein levels can be used to identify ACC subtypes, enabling rapid clinical deployment to guide therapeutic decisions. Our data suggest a model in which ACC-I is driven by MYC signaling through either NOTCH mutations or direct amplification, which in turn suppress P63 signaling observed in ACC-II, producing unique therapeutic vulnerabilities for each subtype., Conclusions: Cooccurrence of multiple actionable protein/pathways alterations in each subtype indicates unique therapeutic vulnerabilities and opportunities for optimal combination therapy for this understudied and heterogeneous disease., (©2020 American Association for Cancer Research.)

Notch receptors participate in a signaling pathway in which ligand-induced proteolysis frees the Notch intracellular domain (NICD), allowing it to translocate to the nucleus, form a transcription complex, and induce target gene expression. Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), splenic marginal zone B-cell lymphoma (SMZL), and distinct subsets of diffuse large B-cell lymphoma (DLBCL) are strongly associated with mutations in the 3' end of NOTCH1 or NOTCH2 that disrupt a proline, glutamic acid, serine, and threonine (PEST) degron domain and stabilize NICD1 and NICD2. By contrast, mutations leading to constitutive Notch activation are rare in primary B-cell neoplasms, suggesting that Notch activation is confined to ligand-rich tumor microenvironments, or that cryptic strong gain-of-function mutations have been missed in prior analyses. To test these ideas, we used immunohistochemical stains to screen a broad range of B-cell tumors for Notch activation. Our analyses reveal that among small B-cell neoplasms, NICD2 is primarily detected in SMZL and is a common feature of both NOTCH2 wild-type and NOTCH2-mutated SMZLs, similar to prior findings with NOTCH1 in CLL/SLL. The greatest NOTCH2 activation was observed in NOTCH2-mutated SMZLs, particularly within splenic marginal zones. By contrast, little evidence of NOTCH2 activation was observed in DLBCL, even in NOTCH2-mutated tumors, suggesting that selective pressure for NOTCH2 activation is mainly confined to low-grade B-cell neoplasms, whereas DLBCLs with NOTCH1 mutations frequently showed evidence of ongoing NOTCH1 activation. These observations have important implications for the pathogenic role of Notch and its therapeutic targeting in B-cell lymphomas., (© 2021 by The American Society of Hematology.)

Adult-onset hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening disease of immune hyperactivation. Unlike pediatric HLH, adult HLH is rarely driven by germline genetic variants. Although numerous precipitating etiologies have been identified, the reason that HLH occurs in only a subset of individuals and how other factors contribute to the disease remains unknown. We hypothesized that clonal hematopoiesis (CH), a state in which somatic mutations in blood cells cause an expanded population of mutant hematopoietic cells and drive an aberrant inflammatory state, could contribute to adult-onset HLH. In a highly annotated cohort of older adults with HLH we found that CH was more prevalent than in control cohorts. Using the adult-onset HLH mouse model in which repeated treatments of the TLR9 agonist, ODN1826, was delivered to the mouse, we observed that macrophages carrying mutations in Tet2, one of the most commonly mutated genes in CH, have an enhanced inflammatory response to TLR9 agonism. Finally, mice carrying Tet2 mutations in the hematopoietic compartment (a common model for CH) displayed an exaggerated response to TLR9 agonism, including worse splenomegaly and anemia. Our data suggest that CH is more common in individuals with adult-onset HLH and can contribute to the pathophysiology of this disease., (© 2020 by The American Society of Hematology.)

Water and lipids are key participants in many biological processes, but there are few non-invasive methods that provide quantification of these components in vivo, and none that can isolate and quantify lipids in the blood. Here we develop a new imaging modality termed shortwave infrared meso-patterned imaging (SWIR-MPI) to provide label-free, non-contact, spatial mapping of water and lipid concentrations in tissue. The method utilizes patterned hyperspectral illumination to target chromophore absorption bands in the 900-1,300 nm wavelength range. We use SWIR-MPI to monitor clinically important physiological processes including edema, inflammation, and tumor lipid heterogeneity in preclinical models. We also show that SWIR-MPI can spatially map blood-lipids in humans, representing an example of non-invasive and contact-free measurements of in vivo blood lipids. Together, these results highlight the potential of SWIR-MPI to enable new capabilities in fundamental studies and clinical monitoring of major conditions including obesity, cancer, and cardiovascular disease.

Notch signaling regulates squamous cell proliferation and differentiation and is frequently disrupted in squamous cell carcinomas, in which Notch is tumor suppressive. Here, we show that conditional activation of Notch in squamous cells activates a context-specific gene expression program through lineage-specific regulatory elements. Among direct Notch target genes are multiple DNA damage response genes, including IER5 , which we show is required for Notch-induced differentiation of squamous carcinoma cells and TERT-immortalized keratinocytes. IER5 is epistatic to PPP2R2A , a gene that encodes the PP2A B55α subunit, which we show interacts with IER5 in cells and in purified systems. Thus, Notch and DNA-damage response pathways converge in squamous cells on common genes that promote differentiation, which may serve to eliminate damaged cells from the proliferative pool. We further propose that crosstalk involving Notch and PP2A enables tuning and integration of Notch signaling with other pathways that regulate squamous differentiation., Competing Interests: LP, ML, TT, JR, CL, WL, CJ, LS, AS, JM, GA No competing interests declared, SB SCB is on the SAB for Erasca, Inc, receives sponsored research funding from Novartis and Erasca, Inc, and is a consultant for IFM therapeutics and Ayala Pharmaceuticals. JA JCA is a consultant for Ayala Pharmaceuticals and for Cellestia, Inc, There is no conflict of interest with the work described in this manuscript., (© 2020, Pan et al.)

Polycomb repressive complex 2 (PRC2) is an epigenetic regulator of gene expression that possesses histone methyltransferase activity. PRC2 trimethylates lysine 27 of histone H3 proteins (H3K27me3) as a chromatin modification associated with repressed transcription of genes frequently involved in cell proliferation or self-renewal. Loss-of-function mutations in the PRC2 core subunit SUZ12 have been identified in a variety of tumors, including malignant peripheral nerve sheath tumors (MPNSTs). To determine the consequences of SUZ12 loss in the pathogenesis of MPNST and other cancers, we used CRISPR-Cas9 to disrupt the open reading frame of each of two orthologous suz12 genes in zebrafish: suz12a and suz12b We generated these knockout alleles in the germline of our previously described p53 (also known as tp53 )- and nf1- deficient zebrafish model of MPNSTs. Loss of suz12 significantly accelerated the onset and increased the penetrance of MPNSTs compared to that in control zebrafish. Moreover, in suz12- deficient zebrafish, we detected additional types of tumors besides MPNSTs, including leukemia with histological characteristics of lymphoid malignancies, soft tissue sarcoma and pancreatic adenocarcinoma, which were not detected in p53/nf1- deficient control fish, and are also contained in the human spectrum of SUZ12 -deficient malignancies identified in the AACR Genie database. The suz12 -knockout tumors displayed reduced or abolished H3K27me3 epigenetic marks and upregulation of gene sets reported to be targeted by PRC2. Thus, these zebrafish lines with inactivation of suz12 in combination with loss of p53/nf1 provide a model of human MPNSTs and multiple other tumor types, which will be useful for mechanistic studies of molecular pathogenesis and targeted therapy with small molecule inhibitors., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Despite decades of clinical use, mechanisms of glucocorticoid resistance are poorly understood. We treated primary murine T lineage acute lymphoblastic leukemias (T-ALLs) with the glucocorticoid dexamethasone (DEX) alone and in combination with the pan-PI3 kinase inhibitor GDC-0941 and observed a robust response to DEX that was modestly enhanced by GDC-0941. Continuous in vivo treatment invariably resulted in outgrowth of drug-resistant clones, ~30% of which showed markedly reduced glucocorticoid receptor (GR) protein expression. A similar proportion of relapsed human T-ALLs also exhibited low GR protein levels. De novo or preexisting mutations in the gene encoding GR (Nr3c1) occurred in relapsed clones derived from multiple independent parental leukemias. CRISPR/Cas9 gene editing confirmed that loss of GR expression confers DEX resistance. Exposing drug-sensitive T-ALLs to DEX in vivo altered transcript levels of multiple genes, and this response was attenuated in relapsed T-ALLs. These data implicate reduced GR protein expression as a frequent cause of glucocorticoid resistance in T-ALL.

Notch pathway signaling is implicated in several human cancers. Aberrant activation and mutations of Notch signaling components are linked to tumor initiation, maintenance, and resistance to cancer therapy. Several strategies, such as monoclonal antibodies against Notch ligands and receptors, as well as small-molecule γ-secretase inhibitors (GSIs), have been developed to interfere with Notch receptor activation at proximal points in the pathway. However, the use of drug-like small molecules to target the downstream mediators of Notch signaling, the Notch transcription activation complex, remains largely unexplored. Here, we report the discovery of an orally active small-molecule inhibitor (termed CB-103) of the Notch transcription activation complex. We show that CB-103 inhibits Notch signaling in primary human T cell acute lymphoblastic leukemia and other Notch-dependent human tumor cell lines, and concomitantly induces cell cycle arrest and apoptosis, thereby impairing proliferation, including in GSI-resistant human tumor cell lines with chromosomal translocations and rearrangements in Notch genes. CB-103 produces Notch loss-of-function phenotypes in flies and mice and inhibits the growth of human breast cancer and leukemia xenografts, notably without causing the dose-limiting intestinal toxicity associated with other Notch inhibitors. Thus, we describe a pharmacological strategy that interferes with Notch signaling by disrupting the Notch transcription complex and shows therapeutic potential for treating Notch-driven cancers., Competing Interests: Competing interest statement: R.L. and F.R. are cofounders of Cellestia Biotech AG, and R.L., M.V., and C.U. are employees of the company.

Greater than 90% of cases of systemic mastocytosis (SM) harbor pathogenic KIT mutations, particularly KIT D816V . Prognostically-significant pathogenic KIT mutations also occur in 30-40% of core binding factor-associated acute myeloid leukemia (CBF-AML), but are uncommonly associated with concurrent SM. By comparison, the occurrence of SM in other myeloid neoplasms bearing pathogenic KIT mutations, particularly those with a chronic course, is poorly understood. Review of clinical next-generation sequencing (NGS) performed at our institutions in patients with known or suspected hematologic malignancies over an 8-year period revealed 64 patients with both a pathogenic KIT mutation detected at one or more timepoints and available bone marrow biopsy materials. Patients with KIT D816V -mutated myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), or overlap MDS/MPN (n = 22) accounted for approximately one-third of our cohort (34%). Comprehensive morphologic and immunophenotypic characterization revealed that nearly all cases (n = 20, 91%) exhibited concurrent SM. In contrast, of the 18 patients (28%) with AML and KIT D816V , only eight (44%) showed evidence of SM at any point in their disease course (p = 0.0021); of these eight, the AML component was characterized as AML with myelodysplasia-related changes (AML-MRC) in all but one instance (n = 7, 87%). Twelve patients (19%) had pathogenic KIT mutations other than p.D816V, all in the setting of AML (CFB-AML, n = 7; AML, not otherwise specified, n = 2; AML-MRC, n = 1; acute promyelocytic leukemia, n = 1); only two of these patients (17%), both with CBF-AML, exhibited concurrent SM. The remaining 12 patients (19%) had SM without evidence of an associated hematological neoplasm (AHN). For nearly one-third of the 30 SM-AHN patients in our cohort (n = 9, 30%), the SM component of their disease was not initially clinicopathologically recognized. We propose that identification of the KIT D816V mutation in patients diagnosed with MDS, MPN, MDS/MPN, or AML-MRC should trigger reflex testing for SM.

Mastermind proteins are required for transcription of Notch target genes, yet the molecular basis for mastermind function remains incompletely understood. Previous work has shown that Notch can induce transcriptional responses by binding to promoters but more often by binding to enhancers, with HES4 and DTX1 as representative mammalian examples of promoter and enhancer responsiveness, respectively. Here, we show that mastermind dependence of the Notch response at these loci is differentially encoded in Jurkat T-cell acute lymphoblastic leukemia (T-ALL) cells. Knockout of Mastermind-like 1 (MAML1) eliminates Notch-responsive activation of both these genes, and reduced target gene expression is accompanied by a decrease in H3K27 acetylation, consistent with the importance of MAML1 for p300 activity. Add-back of MAML1 variants in knockout cells identifies residues 151 to 350 of MAML1 as essential for expression of either Notch-responsive gene. Fusion of the Notch-binding region of MAML1 to the histone acetyltransferase (HAT) domain of p300 rescues expression of HES4 but not DTX1 , suggesting that an additional activity of MAML1 is needed for gene induction at a distance. Together, these studies establish the functional importance of the MAML1 region from residues 151 to 350 for Notch-dependent transcriptional induction and reveal differential requirements for MAML1-dependent recruitment activities at different Notch-responsive loci, highlighting the molecular complexity of Notch-stimulated transcription., (Copyright © 2020 American Society for Microbiology.)

Introduction . Myofibromas are rare tumors of pericytic lineage, typically affecting children, and are sometimes aggressive. A subset of sporadic and familial myofibromas have activating variants in PDGFRB . The relationship of myofibroma and PDGFRB to the NOTCH pathway has not yet been described. Methods . Ten myofibroma cases were sequenced with a targeted panel of 447 genes, including copy number variation and selected fusions. Immunohistochemical analysis of total NOTCH3 and activated NOTCH3 was assessed for all 10 myofibroma cases, and a series of histologic mimics (n = 20). Results . Alterations identified by next-generation sequencing included PDGFRB sequence variants in 8/10 cases (80%), a NOTCH3 variant in 1/10 cases (10%), and a NOTCH2 variant in 1/10 cases (10%). All 10 cases also showed a pattern of low-amplitude (1.5- to 2-fold) copy number alterations including gains in PDGFRB and NOTCH3 . Ten of 10 myofibromas (100%) showed cytoplasmic staining for total NOTCH3 and 9 of 10 cases (90%) showed nuclear staining for activated NOTCH3. Within the control cohort of histologic mimics, 3 of 3 nodular fasciitis cases (100%) were positive for activated and total NOTCH3, and the remaining 17 cases were negative for pan NOTCH3, while 3 of 3 desmoid-type fibromatosis cases (100%) showed patchy weak nuclear staining for activated NOTCH3. Discussion . Our findings suggest a common pathway of PDGFRB/NOTCH3 activation in myofibromas, even in cases that lack PDGFRB sequence variants. These results support the pericytic lineage of myofibroma. Identification of the characteristic genomic alterations or immunohistochemical staining pattern may facilitate a difficult pathologic diagnosis, and support the use of targeted treatments.

Notch receptors participate is a highly conserved signaling pathway that regulates numerous facets of cellular behavior, has protean roles during development and in adult tissue homeostasis, and is frequently dysregulated in human diseases, particularly cancer. These relationships to disease and the ability to modulate Notch signaling at multiple levels have engendered attempts to target Notch therapeutically, but incomplete understanding of the outcomes of Notch activation and on-target toxicity have stymied efforts to date. Using well-controlled experimental systems, we have pursued studies that seek to understand how Notch influences the behavior of different types of cancer cells. Our work suggests that Notch effects are defined by epigenetic landscapes that are "laid out" by upstream pioneer transcription factors, which act to delineate the outcome of Notch activation. These insights define some of the "rules" that govern Notch functions and constitute one step toward bringing safe and effective targeting of Notch to fruition., Competing Interests: Potential Conflicts of Interest: Dr. Aster serves as a consultant for Ayala Pharmaceuticals, Inc., and Cellestia Pharmaceuticals, Inc. The work described herein is independent of his work with these companies., (© 2020 The American Clinical and Climatological Association.)

Canonical Notch signaling relies on regulated proteolysis of the receptor Notch to generate a nuclear effector that induces the transcription of Notch-responsive genes. In higher organisms, one Notch-responsive gene that is activated in many different cell types encodes the Notch-regulated ankyrin repeat protein (NRARP), which acts as a negative feedback regulator of Notch responses. Here, we showed that NRARP inhibited the growth of Notch-dependent T cell acute lymphoblastic leukemia (T-ALL) cell lines and bound directly to the core Notch transcriptional activation complex (NTC), requiring both the transcription factor RBPJ and the Notch intracellular domain (NICD), but not Mastermind-like proteins or DNA. The crystal structure of an NRARP-NICD1-RBPJ-DNA complex, determined to 3.75 Å resolution, revealed that the assembly of NRARP-NICD1-RBPJ complexes relied on simultaneous engagement of RBPJ and NICD1, with the three ankyrin repeats of NRARP extending the Notch1 ankyrin repeat stack. Mutations at the NRARP-NICD1 interface disrupted entry of the proteins into NTCs and abrogated feedback inhibition in Notch signaling assays in cultured cells. Forced expression of NRARP reduced the abundance of NICD in cells, suggesting that NRARP may promote the degradation of NICD. These studies establish the structural basis for NTC engagement by NRARP and provide insights into a critical negative feedback mechanism that regulates Notch signaling., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The interleukin-3 receptor α subunit, CD123, is expressed in many hematologic malignancies including acute myeloid leukemia (AML) and blastic plasmacytoid dendritic cell neoplasm (BPDCN). Tagraxofusp (SL-401) is a CD123-targeted therapy consisting of interleukin-3 fused to a truncated diphtheria toxin payload. Factors influencing response to tagraxofusp other than CD123 expression are largely unknown. We interrogated tagraxofusp resistance in patients and experimental models and found that it was not associated with CD123 loss. Rather, resistant AML and BPDCN cells frequently acquired deficiencies in the diphthamide synthesis pathway, impairing tagraxofusp's ability to ADP-ribosylate cellular targets. Expression of DPH1, encoding a diphthamide pathway enzyme, was reduced by DNA CpG methylation in resistant cells. Treatment with the DNA methyltransferase inhibitor azacitidine restored DPH1 expression and tagraxofusp sensitivity. We also developed a drug-dependent ADP-ribosylation assay in primary cells that correlated with tagraxofusp activity and may represent an additional novel biomarker. As predicted by these results and our observation that resistance also increased mitochondrial apoptotic priming, we found that the combination of tagraxofusp and azacitidine was effective in patient-derived xenografts treated in vivo. These data have important implications for clinical use of tagraxofusp and led to a phase 1 study combining tagraxofusp and azacitidine in myeloid malignancies.