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Abstract: T-prolymphocytic leukemia (T-PLL) is a mature T-cell neoplasm associated with marked chemotherapy resistance and continued poor clinical outcomes. Current treatments, that is, the CD52-antibody alemtuzumab, offer transient responses, with relapses being almost inevitable without consolidating allogeneic transplantation. Recent more detailed concepts of T-PLL's pathobiology fostered the identification of actionable vulnerabilities: (1) altered epigenetics, (2) defective DNA damage responses, (3) aberrant cell-cycle regulation, and (4) deregulated prosurvival pathways, including T-cell receptor and JAK/STAT signaling. To further develop related preclinical therapeutic concepts, we studied inhibitors of histone deacetylases ([H]DACs), B-cell lymphoma 2 (BCL2), cyclin-dependent kinase (CDK), mouse double minute 2 (MDM2), and classical cytostatics, using (1) single-agent and combinatorial compound testing in 20 well-characterized and molecularly profiled primary T-PLL (validated by additional 42 cases) and (2) 2 independent murine models (syngeneic transplants and patient-derived xenografts). Overall, the most efficient/selective single agents and combinations (in vitro and in mice) included cladribine, romidepsin ([H]DAC), venetoclax (BCL2), and/or idasanutlin (MDM2). Cladribine sensitivity correlated with expression of its target RRM2. T-PLL cells revealed low overall apoptotic priming with heterogeneous dependencies on BCL2 proteins. In additional 38 T-cell leukemia/lymphoma lines, TP53 mutations were associated with resistance toward MDM2 inhibitors. P53 of T-PLL cells, predominantly in wild-type configuration, was amenable to MDM2 inhibition, which increased its MDM2-unbound fraction. This facilitated P53 activation and downstream signals (including enhanced accessibility of target-gene chromatin regions), in particular synergy with insults by cladribine. Our data emphasize the therapeutic potential of pharmacologic strategies to reinstate P53-mediated apoptotic responses. The identified efficacies and their synergies provide an informative background on compound and patient selection for trial designs in T-PLL., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Somatic mutations in T cells can cause cancer but also have implications for immunological diseases and cell therapies. The mutation spectrum in nonmalignant T cells is unclear. Here, we examined somatic mutations in CD4 + and CD8 + T cells from 90 patients with hematological and immunological disorders and used T cell receptor (TCR) and single-cell sequencing to link mutations with T cell expansions and phenotypes. CD8 + cells had a higher mutation burden than CD4 + cells. Notably, the biggest variant allele frequency (VAF) of non-synonymous variants was higher than synonymous variants in CD8 + T cells, indicating non-random occurrence. The non-synonymous VAF in CD8 + T cells strongly correlated with the TCR frequency, but not age. We identified mutations in pathways essential for T cell function and often affected lymphoid neoplasia. Single-cell sequencing revealed cytotoxic T EMRA phenotypes of mutated T cells. Our findings suggest that somatic mutations contribute to CD8 + T cell expansions without malignant transformation.

Cancer cells can evade natural killer (NK) cell activity, thereby limiting anti-tumor immunity. To reveal genetic determinants of susceptibility to NK cell activity, we examined interacting NK cells and blood cancer cells using single-cell and genome-scale functional genomics screens. Interaction of NK and cancer cells induced distinct activation and type I interferon (IFN) states in both cell types depending on the cancer cell lineage and molecular phenotype, ranging from more sensitive myeloid to less sensitive B-lymphoid cancers. CRISPR screens in cancer cells uncovered genes regulating sensitivity and resistance to NK cell-mediated killing, including adhesion-related glycoproteins, protein fucosylation genes, and transcriptional regulators, in addition to confirming the importance of antigen presentation and death receptor signaling pathways. CRISPR screens with a single-cell transcriptomic readout provided insight into underlying mechanisms, including regulation of IFN-γ signaling in cancer cells and NK cell activation states. Our findings highlight the diversity of mechanisms influencing NK cell susceptibility across different cancers and provide a resource for NK cell-based therapies., Competing Interests: Declaration of interests M.S. and C.S.M. are authors of a patent application related to antitumor activity of NK cells. C.S.M. is a member of the Scientific Advisory Board of Adicet Bio and also discloses consultant honoraria from Fate Therapeutics, Ionis Pharmaceuticals, FIMECS, Secura Bio, and Oncopeptides and research funding from Sanofi, Merck KGaA/EMD Serono, Arch Oncology, Karyopharm, Nurix, Eisai/H3 Biomedicine, Novartis, BMS, Abcuro, and Springworks. S.M. has received honoraria and research funding from Novartis, Pfizer, and Bristol-Myers Squibb and honoraria from Dren-Bio (not related to this study)., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Prostate cancer (PrCa) genomic heterogeneity causes resistance to therapies such as androgen deprivation. Such heterogeneity can be deciphered in the context of evolutionary principles, but current clinical trials do not include evolution as an essential feature. Whether or not analysis of genomic data in an evolutionary context in primary prostate cancer can provide unique added value in the research and clinical domains remains an open question., Methods: We used novel processing techniques to obtain whole genome data together with 3D anatomic and histomorphologic analysis in two men (GP5 and GP12) with high-risk PrCa undergoing radical prostatectomy. A total of 22 whole genome-sequenced sites (16 primary cancer foci and 6 lymph node metastatic) were analyzed using evolutionary reconstruction tools and spatio-evolutionary models. Probability models were used to trace spatial and chronological origins of the primary tumor and metastases, chart their genetic drivers, and distinguish metastatic and non-metastatic subclones., Results: In patient GP5, CDK12 inactivation was among the first mutations, leading to a PrCa tandem duplicator phenotype and initiating the cancer around age 50, followed by rapid cancer evolution after age 57, and metastasis around age 59, 5 years prior to prostatectomy. In patient GP12, accelerated cancer progression was detected after age 54, and metastasis occurred around age 56, 3 years prior to prostatectomy. Multiple metastasis-originating events were identified in each patient and tracked anatomically. Metastasis from prostate to lymph nodes occurred strictly ipsilaterally in all 12 detected events. In this pilot, metastatic subclone content analysis appears to substantially enhance the identification of key drivers. Evolutionary analysis' potential impact on therapy selection appears positive in these pilot cases., Conclusions: PrCa evolutionary analysis allows tracking of anatomic site of origin, timing of cancer origin and spread, and distinction of metastatic-capable from non-metastatic subclones. This enables better identification of actionable targets for therapy. If extended to larger cohorts, it appears likely that similar analyses could add substantial biological insight and clinically relevant value., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Metatranscriptomics refers to the analysis of the collective microbial transcriptome of a sample. Its increased utilization for the characterization of human-associated microbial communities has enabled the discovery of many disease-state related microbial activities. Here, we review the principles of metatranscriptomics-based analysis of human-associated microbial samples. We describe strengths and weaknesses of popular sample preparation, sequencing, and bioinformatics approaches and summarize strategies for their use. We then discuss how human-associated microbial communities have recently been examined and how their characterization may change. We conclude that metatranscriptomics insights into human microbiotas under health and disease have not only expanded our knowledge on human health, but also opened avenues for rational antimicrobial drug use and disease management., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Autologous split-thickness skin grafts (STSGs) are the standard of care for closure of deep and large burns. However, perforation and extensive fishnet-like expansion of the grafts to achieve greater area wound coverage can lead to treatment failures or esthetically poor healing outcomes and scarring. The purpose of this study was to validate an autologous advanced therapy medicinal product (ATMP)-compliant skin cell suspension and evaluate its efficacy to promote epithelialization., Methods: Cells isolated from a piece of STSG according to ATMP classification requirements were sprayed onto 20 patients during a single operation in a validation study. Comparative evaluation of treatment efficacy was carried out using side-by-side skin graft donor site wounds that were standardized in depth. Firstly, we characterized wound healing transcriptomes at 14 and 21 days from serial wound biopsies in seven patients. Then, side-by-side wounds in four patients were treated with or without the skin cells. The wounds were photographed, clinical outcomes assessed, and the treatment and control wound transcriptomes at 14 days were compared to the untreated wounds' healing transcriptomes., Results: The average cell yield after isolation from the STSG was 2.4 × 10 6 cells/cm 2 with 96 % viability. The product contained mainly keratinocytes and their precursors but also other skin cells such as fibroblasts were present. As compared to vehicle-treated donor site wounds, the wounds treated with cells demonstrated improved epithelialization by both direct comparison and machine learning analysis of the transcriptomes., Conclusions: We showed that rapid and scalable ATMP-classified processing of skin cells is feasible, and application of the skin cells effectively promotes healing and epithelization of donor site wounds., Competing Interests: Conflict of interest The views expressed in this article are those of the authors and do not reflect the official policy or position of the U.S. Army Medical Department, Department of the Army, DOD, or the U.S. Government. All the authors declare no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Metatranscriptomics has revolutionized our ability to explore and understand transcriptional programs in microbial communities. Moreover, it has enabled us to gain deeper and more specific insight into the microbial activities in human gut, respiratory, oral, and vaginal communities. Perhaps the most important contribution of metatranscriptomics arises, however, from the analyses of disease-associated communities. We review the advantages and disadvantages of metatranscriptomics analyses in understanding human health and disease. We focus on human tissues low in microbial biomass and conditions associated with dysbiotic microbiota. We conclude that a more widespread use of metatranscriptomics and increased knowledge on microbe activities will uncover critical interactions between microbes and host in human health and provide diagnostic basis for culturing-independent, direct functional pathogen identification., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Treatment with anaplastic lymphoma kinase (ALK) inhibitors significantly improves outcome for non-small-cell lung cancer (NSCLC) patients with ALK-rearranged tumors. However, clinical resistance typically develops over time and, in the majority of cases, resistance mechanisms are ALK-independent. We generated tumor cell cultures from multiple regions of an ALK-rearranged clinical tumor specimen and deployed functional drug screens to identify modulators of ALK-inhibitor response. This identified a role for PI3Kβ and EGFR inhibition in sensitizing the response regulating resistance to ALK inhibition. Inhibition of ALK elicited activation of EGFR, and subsequent MAPK and PI3K-AKT pathway reactivation. Sensitivity to ALK targeting was enhanced by inhibition or knockdown of PI3Kβ. In ALK-rearranged primary cultures, the combined inhibition of ALK and PI3Kβ prevented the EGFR-mediated ALK-inhibitor resistance, and selectively targeted the cancer cells. The combinatorial effect was seen also in the background of TP53 mutations and in epithelial-to-mesenchymal transformed cells. In conclusion, combinatorial ALK- and PI3Kβ-inhibitor treatment carries promise as a treatment for ALK-rearranged NSCLC., (© 2022 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: Deletions covering the entire or partial JARID2 gene as well as pathogenic single nucleotide variants leading to haploinsufficiency of JARID2 have recently been shown to cause a clinically distinct neurodevelopmental syndrome. Here, we present a previously undescribed partial de novo duplication of the JARID2 gene in a patient displaying features similar to those of patients with JARID2 loss-of-function variants., Case Report: The index patient presents with abnormalities in gross motor skills and speech development as well as neuropsychiatric disorders. The patient has markedly dark infraorbital circles and slightly prominent supraorbital ridges.Whole-genome sequencing and array comparative genomic hybridization revealed a novel disease-causing variant type, a partial tandem duplication of JARID2, covering the exons 1-7. Furthermore, RNA sequencing validated the increased expression of these exons. Expression alterations were also detected in target genes of the PRC2 complex, in which JARID2 acts as an essential member., Conclusion: Our data add to the variety of different pathogenic variants associated with JARID2 neurodevelopmental syndrome., (© 2022 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

T cell large granular lymphocytic leukemia (T-LGLL) is a rare lymphoproliferative disorder of mature, clonally expanded T cells, where somatic-activating STAT3 mutations are common. Although T-LGLL has been described as a chronic T cell response to an antigen, the function of the non-leukemic immune system in this response is largely uncharacterized. Here, by utilizing single-cell RNA and T cell receptor profiling (scRNA+TCRαβ-seq), we show that irrespective of STAT3 mutation status, T-LGLL clonotypes are more cytotoxic and exhausted than healthy reactive clonotypes. In addition, T-LGLL clonotypes show more active cell communication than reactive clones with non-leukemic immune cells via costimulatory cell-cell interactions, monocyte-secreted proinflammatory cytokines, and T-LGLL-clone-secreted IFNγ. Besides the leukemic repertoire, the non-leukemic T cell repertoire in T-LGLL is also more mature, cytotoxic, and clonally restricted than in other cancers and autoimmune disorders. Finally, 72% of the leukemic T-LGLL clonotypes share T cell receptor similarities with their non-leukemic repertoire, linking the leukemic and non-leukemic repertoires together via possible common target antigens. Our results provide a rationale to prioritize therapies that target the entire immune repertoire and not only the T-LGLL clonotype., (© 2022. The Author(s).)

In adult patients, the treatment outcome of acute lymphoblastic leukemia (ALL) remains suboptimal. Here, we used an ex vivo drug testing platform and comprehensive molecular profiling to discover new drug candidates for B-ALL. We analyzed sensitivity of 18 primary B-ALL adult patient samples to 64 drugs in a physiological concentration range. Whole-transcriptome sequencing and publicly available expression data were used to examine gene expression biomarkers for observed drug responses. Apoptotic modulators targeting BCL2 and MDM2 were highly effective. Philadelphia chromosome-negative (Ph-) samples were sensitive to both BCL2/BCL-W/BCL-XL-targeting agent navitoclax and BCL2-selective venetoclax, whereas Ph-positive (Ph+) samples were more sensitive to navitoclax. Expression of BCL2 was downregulated and BCL-W and BCL-XL upregulated in Ph+ ALL compared with Ph- samples, providing elucidation for the observed difference in drug responses. A majority of the samples were sensitive to MDM2 inhibitor idasanutlin. The regulatory protein MDM2 suppresses the function of tumor suppressor p53, leading to impaired apoptosis. In B-ALL, the expression of MDM2 was increased compared with other hematological malignancies. In B-ALL cell lines, a combination of BCL2 and MDM2 inhibitor was synergistic. In summary, antiapoptotic proteins including BCL2 and MDM2 comprise promising targets for future drug studies in B-ALL., (Copyright © 2022 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association.)

We generated ex vivo drug-response and multiomics profiling data for a prospective series of 252 samples from 186 patients with acute myeloid leukemia (AML). A functional precision medicine tumor board (FPMTB) integrated clinical, molecular, and functional data for application in clinical treatment decisions. Actionable drugs were found for 97% of patients with AML, and the recommendations were clinically implemented in 37 relapsed or refractory patients. We report a 59% objective response rate for the individually tailored therapies, including 13 complete responses, as well as bridging five patients with AML to allogeneic hematopoietic stem cell transplantation. Data integration across all cases enabled the identification of drug response biomarkers, such as the association of IL15 overexpression with resistance to FLT3 inhibitors. Integration of molecular profiling and large-scale drug response data across many patients will enable continuous improvement of the FPMTB recommendations, providing a paradigm for individualized implementation of functional precision cancer medicine. SIGNIFICANCE: Oncogenomics data can guide clinical treatment decisions, but often such data are neither actionable nor predictive. Functional ex vivo drug testing contributes significant additional, clinically actionable therapeutic insights for individual patients with AML. Such data can be generated in four days, enabling rapid translation through FPMTB. See related commentary by Letai, p. 290 . This article is highlighted in the In This Issue feature, p. 275 ., (©2021 The Authors; Published by the American Association for Cancer Research.)

Background: Oncolytic viruses are a potent form of active immunotherapy, capable of invoking antitumor T-cell responses. Meanwhile, less is known about their effects on immune checkpoints, the main targets for passive immunotherapy of cancer. T-cell immunoglobulin and mucin domain-3 (TIM-3) is a coinhibitory checkpoint driving T-cell exhaustion in cancer. Here we investigated the effects of oncolytic adenovirus on the TIM-3 checkpoint on tumor-infiltrating immune cells and clinical impact in patients with cancer receiving oncolytic immunotherapy., Methods: Modulation of TIM-3 expression on tumor-infiltrating immune cells was studied preclinically in B16 melanoma following intratumoral treatment with Ad5/3∆24-granulocyte-macrophage colony-stimulating factor oncolytic adenovirus. We conducted a retrospective longitudinal analysis of 15 patients with advanced-stage cancer with tumor-site biopsies before and after oncolytic immunotherapy, treated in the Advanced Therapy Access Program (ISRCTN10141600, April 5, 2011). Following patient stratification with regard to TIM-3 (increase vs decrease in tumors), overall survival and imaging/marker responses were evaluated by log-rank and Fisher's test, while coinhibitory receptors/ligands, transcriptomic changes and tumor-reactive and tumor-infltrating immune cells in biopsies and blood samples were studied by microarray rank-based statistics and immunoassays., Results: Preclinically, TIM-3 + tumor-infiltrating lymphocytes (TILs) in B16 melanoma showed an exhausted phenotype, whereas oncolytic adenovirus treatment significantly reduced the proportion of TIM-3 + TIL subset through recruitment of less-exhausted CD8 + TIL. Decrease of TIM-3 was observed in 60% of patients, which was associated with improved overall survival over TIM-3 increase patients (p=0.004), together with evidence of clinical benefit by imaging and blood analyses. Coinhibitory T-cell receptors and ligands were consistently associated with TIM-3 changes in gene expression data, while core transcriptional exhaustion programs and T-cell dysfunction were enriched in patients with TIM-3 increase, thus identifying patients potentially benefiting from checkpoint blockade. In striking contrast, patients with TIM-3 decrease displayed an acute inflammatory signature, redistribution of tumor-reactive CD8 + lymphocytes and higher influx of CD8 + TIL into tumors, which were associated with the longest overall survival, suggesting benefit from active immunotherapy., Conclusions: Our results indicate a key role for the TIM-3 immune checkpoint in oncolytic adenoviral immunotherapy. Moreover, our results identify TIM-3 as a potential biomarker for oncolytic adenoviruses and create rationale for combination with passive immunotherapy for a subset of patients., Competing Interests: Competing interests: OH and AH hold shares in Targovax ASA and in TILT Biotherapeutics, and AH is an employee in TILT Biotherapeutics. The other authors declare that they have no competing financial interests., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

Renal cell carcinoma (RCC) is considered as an immunogenic cancer. Because not all patients respond to current immunotherapies, we aimed to investigate the immunological heterogeneity of RCC tumors. We analyzedthe immunophenotype of the circulating, tumor, and matching adjacent healthy kidney immune cells from 52 nephrectomy patients with multi-parameter flow cytometry. Additionally, we studied the transcriptomic and mutation profiles of 20 clear cell RCC (ccRCC) tumors with bulk RNA sequencing and a customized pan-cancer gene panel. The tumor samples clustered into two distinct subgroups defined by the abundance of intratumoral CD3+ T cells (CD3 high , 25/52) and NK cells (NK high , 27/52). CD3 high tumors had an overall higher frequency of tumor infiltrating lymphocytes and PD-1 expression on the CD8+ T cells compared to NK high tumors. The tumor infiltrating T and NK cells had significantly elevated expression levels of LAG-3, PD-1, and HLA-DR compared to the circulating immune cells. Transcriptomic analysis revealed increased immune signaling (IFN-γ, TNF-α via NF-κB, and T cell receptor signaling) and kidney metabolism pathways in the CD3 high subgroup. Genomic analysis confirmed the typical ccRCC mutation profile including VHL, PBRM1 , and SETD2 mutations, and revealed PBRM1 as a uniquely mutated gene in the CD3 high subgroup. Approximately half of the RCC tumors have a high infiltration of NK cells associated with a lower number of tumor infiltrating lymphocytes, lower PD-1 expression, a distinct transcriptomic and mutation profile, providing insights to the immunological heterogeneity of RCC which may impact treatment responses to immunological therapies., Competing Interests: SM has received honoraria and research funding from Bristol Myers Squibb, Novartis, and Pfizer outside the submitted work. PJ has received funding from Elypta Ab. AK is currently employed by Novartis. OB has received honoraria from Novartis and Sanofi outside the submitted work. The author(s) declare that there are no other conflicts of interest., (© 2022 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Background: Although many pathological changes have been associated with ischemic heart disease (IHD), molecular-level alterations specific to the ischemic myocardium and their potential to reflect disease severity or therapeutic outcome remain unclear. Currently, diagnosis occurs relatively late and evaluating disease severity is largely based on clinical symptoms, various imaging modalities, or the determination of risk factors. This study aims to identify IHD-associated signature RNAs from the atrial myocardium and evaluate their ability to reflect disease severity or cardiac surgery outcomes. Methods and Results: We collected right atrial appendage (RAA) biopsies from 40 patients with invasive coronary angiography (ICA)-positive IHD undergoing coronary artery bypass surgery and from 8 patients ICA-negative for IHD (non-IHD) undergoing valvular surgery. Following RNA sequencing, RAA transcriptomes were analyzed against 429 donors from the GTEx project without cardiac disease. The IHD transcriptome was characterized by repressed RNA expression in pathways for cell-cell contacts and mitochondrial dysfunction. Increased expressions of the CSRNP3, FUT10, SHD, NAV2-AS4 , and hsa-mir-181 genes resulted in significance with the complexity of coronary artery obstructions or correlated with a functional cardiac benefit from bypass surgery. Conclusions: Our results provide an atrial myocardium-focused insight into IHD signature RNAs. The specific gene expression changes characterized here, pave the way for future disease mechanism-based identification of biomarkers for early detection and treatment of IHD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mulari, Eskin, Lampinen, Nummi, Nieminen, Teittinen, Ojala, Kankainen, Vento, Laurikka, Kupari, Harjula, Tuncbag and Kankuri.)

Although the development of hematopoietic stem cells (HSC) has been studied in great detail, their heterogeneity and relationships to different cell lineages remain incompletely understood. Moreover, the role of Vascular Adhesion Protein-1 in bone marrow hematopoiesis has remained unknown. Here we show that VAP-1, an adhesin and a primary amine oxidase producing hydrogen peroxide, is expressed on a subset of human HSC and bone marrow vasculature forming a hematogenic niche. Bulk and single-cell RNAseq analyses reveal that VAP-1 + HSC represent a transcriptionally unique small subset of differentiated and proliferating HSC, while VAP-1 - HSC are the most primitive HSC. VAP-1 generated hydrogen peroxide acts via the p53 signaling pathway to regulate HSC proliferation. HSC expansion and differentiation into colony-forming units are enhanced by inhibition of VAP-1. Contribution of VAP-1 to HSC proliferation was confirmed with mice deficient of VAP-1, mice expressing mutated VAP-1 and using an enzyme inhibitor. In conclusion, VAP-1 expression allows the characterization and prospective isolation of a new subset of human HSC. Since VAP-1 serves as a check point-like inhibitor in HSC differentiation, the use of VAP-1 inhibitors enables the expansion of HSC., (© 2021. The Author(s).)

Large granular lymphocyte leukemia (LGLL) is characterized by somatic gain-of-function STAT3 mutations. However, the functional effects of STAT3 mutations on primary LGLL cells have not been studied in detail. In this study, we show that CD8+ T cells isolated from STAT3 mutated LGLL patients have high protein levels of epigenetic regulators, such as DNMT1, and are characterized by global hypermethylation. Correspondingly, treatment of healthy CD8+ T cells with IL-6, IL-15, and/or MCP-1 cytokines resulted in STAT3 activation, increased DNMT1, EZH2, c-MYC, l-MYC, MAX, and NFκB levels, increased DNA methylation, and increased oxidative stress. Similar results were discovered in KAI3 NK cells overexpressing gain-of-function STAT3 Y640F and STAT3 G618R mutants compared to KAI3 NK cells overexpressing STAT3 WT . Our results also confirm that STAT3 forms a direct complex with DNMT1, EZH2, and HDAC1. In STAT3 mutated LGLL cells, DNA methyltransferase (DNMT) inhibitor azacitidine abrogated the activation of STAT3 via restored SHP1 expression. In conclusion, STAT3 mutations cause DNA hypermethylation resulting in sensitivity to DNMT inhibitors, which could be considered as a novel treatment option for LGLL patients with resistance to standard treatments., (© 2021. The Author(s).)

The BCR-ABL1 fusion gene, which causes aberrant kinase activity and uncontrolled cell proliferation, is the hallmark of chronic myeloid leukemia (CML). The development of tyrosine kinase inhibitors (TKI) that target the BCR-ABL oncoprotein has led to dramatic improvement in CML management. However, some challenges remain to be addressed in the TKI era, including patient stratification and the selection of frontline TKIs and CML progression. Additionally, with the emerging goal of treatment-free remission (TFR) in CML management, biomarkers that predict the outcomes of stopping TKI remain to be identified. Notably, recent reports have revealed the power of genome screening in understanding the role of genome aberrations other than BCR-ABL1 in CML pathogenesis. These studies have discovered the presence of disease-phase specific mutations and linked certain mutations to inferior responses to TKI treatment and CML progression. A personalized approach that incorporates genetic data in tailoring treatment strategies has been successfully implemented in acute leukemia, and it represents a promising approach for the management of high-risk CML patients. In this article, we will review current knowledge about the mutational profile in different phases of CML as well as patterns of mutational dynamics in patients having different outcomes. We highlight the effects of somatic mutations involving certain genes (e.g. epigenetic modifiers) on the outcomes of TKI treatment. We also discuss the potential value of incorporating genetic data in treatment decisions and the routine care of CML patients as a future direction for optimizing CML management.

Cancer-associated fibroblasts (CAFs) are known to increase tumor growth and to stimulate invasion and metastasis. Increasing evidence suggests that CAFs mediate response to various treatments. HNSCC cell lines were co-cultured with their patient-matched CAFs in 2D and 3D in vitro models, and the tumor cell gene expression profiles were investigated by cDNA microarray and qRT-PCR. The mRNA expression of eight candidate genes was examined in tumor biopsies from 32 HNSCC patients and in five biopsies from normal oral tissue. Differences in overall survival (OS) were tested with Kaplan-Meier long-rank analysis. Thirteen protein coding genes were found to be differentially expressed in tumor cells co-cultured with CAFs in 2D and 81 in 3D when compared to tumor cells cultured without CAFs. Six of these genes were upregulated both in 2D and 3D ( POSTN, GREM1, BGN, COL1A2, COL6A3 , and COL1A1 ). Moreover, two genes upregulated in 3D, MMP9 and FMOD, were significantly associated with the OS. In conclusion, we demonstrated in vitro that CAF-derived signals alter the tumor cell expression of multiple genes, several of which are associated with differentiation, epithelial-to-mesenchymal transition (EMT) phenotype, and metastasis. Moreover, six of the most highly upregulated genes were found to be overexpressed in tumor tissue compared to normal tissue.

The oncogenic protein Bcr-Abl has two major isoforms, p190 Bcr-Abl and p210 Bcr-Abl . While p210 Bcr-Abl is the hallmark of chronic myeloid leukemia (CML), p190 Bcr-Abl occurs in the majority of Philadelphia-positive acute lymphoblastic leukemia (Ph + ALL) patients. In CML, p190 Bcr-Abl occurs in a minority of patients associating with distinct hematological features and inferior outcomes, yet the pathogenic role of p190 Bcr-Abl and potential targeting therapies are largely uncharacterized. We employed next generation sequencing, phospho-proteomic profiling, and drug sensitivity testing to characterize p190 Bcr-Abl in CML and hematopoietic progenitor cell line models (Ba/f3 and HPC-LSK). p190 Bcr-Abl CML patients demonstrated poor response to imatinib and frequent mutations in epigenetic modifiers genes. In contrast with p210 Bcr-Abl , p190 Bcr-Abl exhibited specific transcriptional upregulation of interferon, interleukin-1 receptor, and P53 signaling pathways, associated with hyperphosphorylation of relevant signaling molecules including JAK1/STAT1 and PAK1 in addition to Src hyperphosphorylation. Comparable to p190 Bcr-Abl CML patients, p190 Bcr-Abl cell lines demonstrated similar transcriptional and phospho-signaling signatures. With the drug sensitivity screening we identified targeted drugs with specific activity in p190 Bcr-Abl cell lines including IAP-, PAK1-, and Src inhibitors and glucocorticoids. Our results provide novel insights into the mechanisms underlying the distinct features of p190 Bcr-Abl CML and promising therapeutic targets for this high-risk patient group.

The prevalence and functional impact of somatic mutations in nonleukemic T cells is not well characterized, although clonal T-cell expansions are common. In immune-mediated aplastic anemia (AA), cytotoxic T-cell expansions are shown to participate in disease pathogenesis. We investigated the mutation profiles of T cells in AA by a custom panel of 2533 genes. We sequenced CD4+ and CD8+ T cells of 24 AA patients and compared the results to 20 healthy controls and whole-exome sequencing of 37 patients with AA. Somatic variants were common both in patients and healthy controls but enriched to AA patients' CD8+ T cells, which accumulated most mutations on JAK-STAT and MAPK pathways. Mutation burden was associated with CD8+ T-cell clonality, assessed by T-cell receptor beta sequencing. To understand the effect of mutations, we performed single-cell sequencing of AA patients carrying STAT3 or other mutations in CD8+ T cells. STAT3 mutated clone was cytotoxic, clearly distinguishable from other CD8+ T cells, and attenuated by successful immunosuppressive treatment. Our results suggest that somatic mutations in T cells are common, associate with clonality, and can alter T-cell phenotype, warranting further investigation of their role in the pathogenesis of AA.

Blast-phase chronic myeloid leukemia (BP-CML) is associated with additional chromosomal aberrations, RUNX1 mutations being one of the most common. Tyrosine kinase inhibitor therapy has only limited efficacy in BP-CML, and characterization of more defined molecular subtypes is warranted in order to design better treatment modalities for this poor prognosis patient group. Using whole-exome and RNA sequencing we demonstrate that PHF6 and BCORL1 mutations, IKZF1 deletions, and AID/RAG-mediated rearrangements are enriched in RUNX1 mut BP-CML leading to typical mutational signature. On transcriptional level interferon and TNF signaling were deregulated in primary RUNX1 mut CML cells and stem cell and B-lymphoid factors upregulated giving a rise to distinct phenotype. This was accompanied with the sensitivity of RUNX1 mut blasts to CD19-CAR T cells in ex vivo assays. High-throughput drug sensitivity and resistance testing revealed leukemia cells from RUNX1 mut patients to be highly responsive for mTOR-, BCL2-, and VEGFR inhibitors and glucocorticoids. These findings were further investigated and confirmed in CRISPR/Cas9-edited homozygous RUNX1 -/- and heterozygous RUNX1 -/mut BCR-ABL positive cell lines. Overall, our study provides insights into the pathogenic role of RUNX1 mutations and highlights personalized targeted therapy and CAR T-cell immunotherapy as potentially promising strategies for treating RUNX1 mut BP-CML patients.

Linked-read sequencing was developed to aid the detection of large structural variants (SVs) from short-read sequencing efforts. We performed a systematic evaluation to determine if linked-read exome sequencing provides more comprehensive and clinically relevant information than whole-exome sequencing (WES) when applied to the same set of multiple myeloma patient samples. We report that linked-read sequencing detected a higher number of SVs (n = 18,455) than WES (n = 4065). However, linked-read predictions were dominated by inversions (92.4%), leading to poor detection of other types of SVs. In contrast, WES detected 56.3% deletions, 32.6% insertions, 6.7% translocations, 3.3% duplications and 1.2% inversions. Surprisingly, the quantitative performance assessment suggested a higher performance for WES (AUC = 0.791) compared to linked-read sequencing (AUC = 0.766) for detecting clinically validated cytogenetic alterations. We also found that linked-read sequencing detected more short variants (n = 704) compared to WES (n = 109). WES detected somatic mutations in all MM-related genes while linked-read sequencing failed to detect certain mutations. The comparison of somatic mutations detected using linked-read, WES and RNA-seq revealed that WES and RNA-seq detected more mutations than linked-read sequencing. These data indicate that WES outperforms and is more efficient than linked-read sequencing for detecting clinically relevant SVs and MM-specific short variants.

CD73 is an important ectoenzyme responsible for the production of extracellular adenosine. It is involved in regulating inflammatory responses and cell migration and is overexpressed in various cancers. The functions of CD73 in blood endothelial cells are understood in detail, but its role on afferent lymphatics remains unknown. Moreover, anti-CD73 antibodies are now used in multiple clinical cancer trials, but their effects on different endothelial cell types have not been studied. This study reveals that a previously unknown role of CD73 on afferent lymphatics is to dampen immune responses. Knocking it out or suppressing it by siRNA leads to the upregulation of inflammation-associated genes on lymphatic endothelial cells and a more pro-inflammatory phenotype of interacting dendritic cells in vitro and in vivo. In striking contrast, anti-CD73 antibodies had only negligible effects on the gene expression of lymphatic- and blood-endothelial cells. Our data thus reveal new functions of lymphatic CD73 and indicate a low likelihood of endothelial cell-related adverse effects by CD73 targeting therapeutic antibodies., (© 2020 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Background: Inflammatory upper airway diseases cause significant morbidity. They include phenotypes with different treatment; allergic or non-allergic rhinitis (AR, nAR), and chronic rhinosinusitis with or without nasal polyps (CRSwNP, CRSsNP). In clinical practice, these phenotypes are often difficult to distinguish and may overlap., Objective: To evaluate if hierarchical clustering can be used to distinguish these phenotypes based on the presence of nasal polyps, off-seasonal allergic symptoms, and self-reported background characteristics - e.g. atopic dermatitis (AD); and to further analyse the obtained clusters., Methods: We studied a random sample of 74 CRS (chronic rhinosinusitis) patients, and a control group of 80 subjects without CRS with/without AR (tertiary hospitals, 2006-2012). All underwent interview and nasal examination, and filled a questionnaire. Variables regarding demographics, off-seasonal symptoms, and clinical findings were collected. Hierarchical clustering was performed, the obtained clusters were cross-tabulated and analysed., Results: Four clusters were identified; 1: "Severe symptoms and CRSwNP" (n = 29), 2: "Asymptomatic AR and controls" (n = 39), 3: "Moderate symptoms and CRSsNP" (n = 36), and 4: "Symptomatic and AD" (n = 50). Cluster 1 had most sinonasal symptoms, cluster 3 had a high prevalence of facial pain. The presence of AR did not distinguish CRS groups. Of the AR subjects, 51 % belonged to cluster 4, where AR with off-seasonal airway symptoms and AD predominated., Conclusions: Hierarchical clustering can be used to distinguish inflammatory upper airway disease phenotypes. The AR phenotype was subdivided by the presence of AD. Adult AR+ AD patients could benefit from active clinical care of the upper airways also off-season.

Common variable immunodeficiency and other late-onset immunodeficiencies often co-manifest with autoimmunity and lymphoproliferation. The pathogenesis of most cases is elusive, as only a minor subset harbors known monogenic germline causes. The involvement of both B and T cells is however implicated. To study whether somatic mutations in CD4+ and CD8+ T cells associate with immunodeficiency, we recruited 17 patients and 21 healthy controls. Eight patients had late-onset common variable immunodeficiency and nine patients other immunodeficiency and/or severe autoimmunity. In total, autoimmunity occurred in 94% and lymphoproliferation in 65%. We performed deep sequencing of 2533 immune-associated genes from CD4+ and CD8+ cells. Deep T-cell receptor beta sequencing was used to characterize CD4+ and CD8+ T-cell receptor repertoires. The prevalence of somatic mutations was 65% in all immunodeficiency patients, 75% in common variable immunodeficiency and 48% in controls. Clonal hematopoiesis-associated variants in both CD4+ and CD8+ cells occurred in 24% of immunodeficiency patients. Results demonstrated mutations in known tumor suppressors, oncogenes, and genes that are critical for immune- and proliferative functions, such as STAT5B (two patients), C5AR1 (two patients), KRAS (one patient), and NOD2 (one patient). Additionally, as a marker of T-cell receptor repertoire perturbation, common variable immunodeficiency patients harbored increased frequencies of clones with identical complementarity determining region 3 sequences despite unique nucleotide sequences when compared to controls. In conclusion, somatic mutations in genes implicated for autoimmunity and lymphoproliferation are common in CD4+ and CD8+ cells of patients with immunodeficiency. They may contribute to immune dysregulation in a subset of immunodeficiency patients.

Rheumatoid arthritis (RA) is a complex autoimmune disease targeting synovial joints. Traditionally, RA is divided into seropositive (SP) and seronegative (SN) disease forms, the latter consisting of an array of unrelated diseases with joint involvement. Recently, we described a severe form of SN-RA that associates with characteristic joint destruction. Here, we sought biological characteristics to differentiate this rare but aggressive anti-citrullinated peptide antibody-negative destructive RA (CND-RA) from early seropositive (SP-RA) and seronegative rheumatoid arthritis (SN-RA). We also aimed to study cytotoxic CD8+ lymphocytes in autoimmune arthritis. CND-RA, SP-RA and SN-RA were compared to healthy controls to reveal differences in T-cell receptor beta (TCRβ) repertoire, cytokine levels and autoantibody repertoires. Whole-exome sequencing (WES) followed by single-cell RNA-sequencing (sc-RNA-seq) was performed to study somatic mutations in a clonally expanded CD8+ lymphocyte population in an index patient. A unique TCRβ signature was detected in CND-RA patients. In addition, CND-RA patients expressed higher levels of the bone destruction-associated TNFSF14 cytokine. Blood IgG repertoire from CND-RA patients recognized fewer endogenous proteins than SP-RA patients' repertoires. Using WES, we detected a stable mutation profile in the clonally expanded CD8+ T-cell population characterized by cytotoxic gene expression signature discovered by sc-RNA-sequencing. Our results identify CND-RA as an independent RA subset and reveal a CND-RA specific TCR signature in the CD8+ lymphocytes. Improved classification of seronegative RA patients underlines the heterogeneity of RA and also, facilitates development of improved therapeutic options for the treatment resistant patients., (Copyright © 2020 Kelkka, Savola, Bhattacharya, Huuhtanen, Lönnberg, Kankainen, Paalanen, Tyster, Lepistö, Ellonen, Smolander, Eldfors, Yadav, Khan, Koivuniemi, Sjöwall, Elo, Lähdesmäki, Maeda, Nishikawa, Leirisalo-Repo, Sokka-Isler and Mustjoki.)

Genome wide association studies (GWASs) have revealed several airway disease-associated risk loci. Their role in the onset of asthma, allergic rhinitis (AR) or chronic rhinosinusitis (CRS), however, is not yet fully understood. The aim of this review is to evaluate the airway relevance of loci and genes identified in GWAS studies. GWASs were searched from databases, and a list of loci associating significantly (p < 10 -8 ) with asthma, AR and CRS was created. This yielded a total of 267 significantly asthma/AR-associated loci from 31 GWASs. No significant CRS -associated loci were found in this search. A total of 170 protein coding genes were connected to these loci. Of these, 76/170 (44%) showed bronchial epithelial protein expression in stained microscopic figures of Human Protein Atlas (HPA), and 61/170 (36%) had a literature report of having airway epithelial function. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation analyses were performed, and 19 functional protein categories were found as significantly (p < 0.05) enriched among these genes. These were related to cytokine production, cell activation and adaptive immune response, and all were strongly connected in network analysis. We also identified 15 protein pathways that were significantly (p < 0.05) enriched in these genes, related to T-helper cell differentiation, virus infection, JAK-STAT signaling pathway, and asthma. A third of GWAS-level risk loci genes of asthma or AR seemed to have airway epithelial functions according to our database and literature searches. In addition, many of the risk loci genes were immunity related. Some risk loci genes also related to metabolism, neuro-musculoskeletal or other functions. Functions overlapped and formed a strong network in our pathway analyses and are worth future studies of biomarker and therapeutics., Competing Interests: Competing interestsSTS has acted as paid consultant for Mylan Laboratories Ltd., Biomedical systems Ltd., Roche Products Ltd., and Sanofi S.A. All other authors declare no conflicts of interest., (© The Author(s) 2020.)

Understanding factors that shape the immune landscape across hematological malignancies is essential for immunotherapy development. We integrated over 8,000 transcriptomes and 2,000 samples with multilevel genomics of hematological cancers to investigate how immunological features are linked to cancer subtypes, genetic and epigenetic alterations, and patient survival, and validated key findings experimentally. Infiltration of cytotoxic lymphocytes was associated with TP53 and myelodysplasia-related changes in acute myeloid leukemia, and activated B cell-like phenotype and interferon-γ response in lymphoma. CIITA methylation regulating antigen presentation, cancer type-specific immune checkpoints, such as VISTA in myeloid malignancies, and variation in cancer antigen expression further contributed to immune heterogeneity and predicted survival. Our study provides a resource linking immunology with cancer subtypes and genomics in hematological malignancies., Competing Interests: Declaration of Interests S.M. has received honoraria and research funding from Novartis, Pfizer, and Bristol-Myers Squibb, and C.H. received funding from Celgene, Novartis, Oncopeptides, Orion Pharma, and the Innovative Medicines Initiative project HARMONY. The remaining authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Graft versus host disease (GvHD) is the main complication of allogeneic hematopoietic stem cell transplantation (HSCT). Here we report studies of a patient with chronic GvHD (cGvHD) carrying persistent CD4 + T cell clonal expansion harboring somatic mTOR, NFKB2, and TLR2 mutations. In the screening cohort (n = 134), we detect the mTOR P2229R kinase domain mutation in two additional cGvHD patients, but not in healthy or HSCT patients without cGvHD. Functional analyses of the mTOR mutation indicate a gain-of-function alteration and activation of both mTORC1 and mTORC2 signaling pathways, leading to increased cell proliferation and decreased apoptosis. Single-cell RNA sequencing and real-time impedance measurements support increased cytotoxicity of mutated CD4 + T cells. High throughput drug-sensitivity testing suggests that mutations induce resistance to mTOR inhibitors, but increase sensitivity for HSP90 inhibitors. Our findings imply that somatic mutations may contribute to aberrant T cell proliferations and persistent immune activation in cGvHD, thereby paving the way for targeted therapies.

Chimeric antigen receptor (CAR) T-cell therapy has proven effective in relapsed and refractory B-cell malignancies, but resistance and relapses still occur. Better understanding of mechanisms influencing CAR T-cell cytotoxicity and the potential for modulation using small-molecule drugs could improve current immunotherapies. Here, we systematically investigated druggable mechanisms of CAR T-cell cytotoxicity using >500 small-molecule drugs and genome-scale CRISPR-Cas9 loss-of-function screens. We identified several tyrosine kinase inhibitors that inhibit CAR T-cell cytotoxicity by impairing T-cell signaling transcriptional activity. In contrast, the apoptotic modulator drugs SMAC mimetics sensitized B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma cells to anti-CD19 CAR T cells. CRISPR screens identified death receptor signaling through FADD and TNFRSF10B (TRAIL-R2) as a key mediator of CAR T-cell cytotoxicity and elucidated the RIPK1-dependent mechanism of sensitization by SMAC mimetics. Death receptor expression varied across genetic subtypes of B-cell malignancies, suggesting a link between mechanisms of CAR T-cell cytotoxicity and cancer genetics. These results implicate death receptor signaling as an important mediator of cancer cell sensitivity to CAR T-cell cytotoxicity, with potential for pharmacological targeting to enhance cancer immunotherapy. The screening data provide a resource of immunomodulatory properties of cancer drugs and genetic mechanisms influencing CAR T-cell cytotoxicity., (© 2020 by The American Society of Hematology.)

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm accounting for ∼15% of all leukemia. Progress of the disease from an indolent chronic phase to the more aggressive accelerated phase or blast phase (BP) occurs in a minority of cases and is associated with an accumulation of somatic mutations. We performed genetic profiling of 85 samples and transcriptome profiling of 12 samples from 59 CML patients. We identified recurrent somatic mutations in ABL1 (37%), ASXL1 (26%), RUNX1 (16%), and BCOR (16%) in the BP and observed that mutation signatures in the BP resembled those of acute myeloid leukemia (AML). We found that mutation load differed between the indolent and aggressive phases and that nonoptimal responders had more nonsilent mutations than did optimal responders at the time of diagnosis, as well as in follow-up. Using RNA sequencing, we identified other than BCR-ABL1 cancer-associated hybrid genes in 6 of the 7 BP samples. Uncovered expression alterations were in turn associated with mechanisms and pathways that could be targeted in CML management and by which somatic alterations may emerge in CML. Last, we showed the value of genetic data in CML management in a personalized medicine setting., (© 2020 by The American Society of Hematology.)

Many sarcoidosis-associating immunological genes have been shown to be shared between other immune-mediated diseases. In Finnish sarcoidosis patients, good prognosis subjects more commonly have HLA-DRB1 * 03:01 and/or HLA-DRB1 * 04:01-DPB1 * 04:01 haplotype, but no marker for persistent disease have been found. The objective was to further pinpoint genetic differences between prognosis subgroups in relation to the HLA markers. Whole-exome sequencing was conducted for 72 patients selected based on disease activity (resolved disease, n = 36; persistent disease, n = 36). Both groups were further divided by the HLA markers (one/both markers, n = 18; neither of the markers, n = 18). The Finnish exome data from the Genome Aggregation Database was used as a control population in the WES sample. Statistical analyses included single-variant analysis for common variants and gene level analysis for rare variants. We attempted to replicate associated variants in 181 Finnish sarcoidosis patients and 150 controls. An association was found in chromosome 1p36.21 (AADACL3 and C1orf158), which has recently been associated with sarcoidosis in another WES study. In our study, variations in these genes were associated with resolved disease (AADACL3, p = 0.0001 and p = 0.0003; C1orf158, p = 7.03E-05). Another interesting chromosomal region also peaked, Leucocyte Receptor Complex in 19q13.42, but the association diminished in the replication sample. In conclusion, this WES study supports the previously found association in the region 1p36.21. Furthermore, a novel to sarcoidosis region was found, but additional studies are warranted to verify this association., (Copyright © 2019 Lahtela, Kankainen, Sinisalo, Selroos and Lokki.)

Background: Ischemia-reperfusion injury may compromise the short-term and long-term prognosis after heart transplantation. Experimental studies show that simvastatin administered to the organ donor is vasculoprotective and inhibits cardiac allograft ischemia-reperfusion injury., Methods: Eighty-four multiorgan donors were randomly assigned to receive 80 mg of simvastatin (42 donors) via nasogastric tube after declaration of brain death and upon acceptance as a cardiac donor, or to receive no simvastatin (42 donors). The primary efficacy end point was postoperative plasma troponin T and I levels during the first 24 hours after heart transplantation. Secondary end points included postoperative hemodynamics, inflammation, allograft function, rejections and rejection treatments, and mortality.   Results: Organ donor simvastatin treatment significantly reduced the heart recipient plasma levels of troponin T by 34% (14 900 ± 12 100 ng/L to 9800 ± 7900 ng/L, P=0.047), and troponin I by 40% (171 000 ± 151 000 ng/L to 103 000 ± 109 000 ng/L, P=0.023) at 6 hours after reperfusion, the levels of NT-proBNP (N-terminal pro-B-type natriuretic peptide) by 36% (32 800 ± 24 300 ng/L to 20 900 ± 15 900 ng/L; P=0.011) at 1 week, and the number of rejection treatments with hemodynamic compromise by 53% within the first 30 days (P=0.046). Donor simvastatin treatment did not affect donor lipid levels but was associated with a specific transplant myocardial biopsy gene expression profile, and a decrease in recipient postoperative plasma levels of CXCL10 (C-X-C motif chemokine 10), interleukin-1α, placental growth factor, and platelet-derived growth factor-BB. Postoperative hemodynamics, biopsy-proven acute rejections, and mortality were similar. No adverse effects were seen in recipients receiving noncardiac solid organ transplants from simvastatin-treated donors., Conclusions: Donor simvastatin treatment reduces biomarkers of myocardial injury after heart transplantation, and-also considering its documented general safety profile-may be used as a novel, safe, and inexpensive adjunct therapy in multiorgan donation., Clinical Trial Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01160978.

Cancer cells with heterogeneous mutation landscapes and extensive functional redundancy easily develop resistance to monotherapies by emerging activation of compensating or bypassing pathways. To achieve more effective and sustained clinical responses, synergistic interactions of multiple druggable targets that inhibit redundant cancer survival pathways are often required. Here, we report a systematic polypharmacology strategy to predict, test, and understand the selective drug combinations for MDA-MB-231 triple-negative breast cancer cells. We started by applying our network pharmacology model to predict synergistic drug combinations. Next, by utilizing kinome-wide drug-target profiles and gene expression data, we pinpointed a synergistic target interaction between Aurora B and ZAK kinase inhibition that led to enhanced growth inhibition and cytotoxicity, as validated by combinatorial siRNA, CRISPR/Cas9, and drug combination experiments. The mechanism of such a context-specific target interaction was elucidated using a dynamic simulation of MDA-MB-231 signaling network, suggesting a cross-talk between p53 and p38 pathways. Our results demonstrate the potential of polypharmacological modeling to systematically interrogate target interactions that may lead to clinically actionable and personalized treatment options., Competing Interests: Competing interestsThe authors declare no competing interests.

Background: Tamoxifen treatment of estrogen receptor (ER)-positive breast cancer reduces mortality by 31%. However, over half of advanced ER-positive breast cancers are intrinsically resistant to tamoxifen and about 40% will acquire the resistance during the treatment., Methods: In order to explore mechanisms underlying endocrine therapy resistance in breast cancer and to identify new therapeutic opportunities, we created tamoxifen-resistant breast cancer cell lines that represent the luminal A or the luminal B. Gene expression patterns revealed by RNA-sequencing in seven tamoxifen-resistant variants were compared with their isogenic parental cells. We further examined those transcriptomic alterations in a publicly available patient cohort., Results: We show that tamoxifen resistance cannot simply be explained by altered expression of individual genes, common mechanism across all resistant variants, or the appearance of new fusion genes. Instead, the resistant cell lines shared altered gene expression patterns associated with cell cycle, protein modification and metabolism, especially with the cholesterol pathway. In the tamoxifen-resistant T-47D cell variants we observed a striking increase of neutral lipids in lipid droplets as well as an accumulation of free cholesterol in the lysosomes. Tamoxifen-resistant cells were also less prone to lysosomal membrane permeabilization (LMP) and not vulnerable to compounds targeting the lipid metabolism. However, the cells were sensitive to disulfiram, LCS-1, and dasatinib., Conclusion: Altogether, our findings highlight a major role of LMP prevention in tamoxifen resistance, and suggest novel drug vulnerabilities associated with this phenotype.

Aggressive natural killer-cell (NK-cell) leukemia (ANKL) is an extremely aggressive malignancy with dismal prognosis and lack of targeted therapies. Here, we elucidate the molecular pathogenesis of ANKL using a combination of genomic and drug sensitivity profiling. We study 14 ANKL patients using whole-exome sequencing (WES) and identify mutations in STAT3 (21%) and RAS-MAPK pathway genes (21%) as well as in DDX3X (29%) and epigenetic modifiers (50%). Additional alterations include JAK-STAT copy gains and tyrosine phosphatase mutations, which we show recurrent also in extranodal NK/T-cell lymphoma, nasal type (NKTCL) through integration of public genomic data. Drug sensitivity profiling further demonstrates the role of the JAK-STAT pathway in the pathogenesis of NK-cell malignancies, identifying NK cells to be highly sensitive to JAK and BCL2 inhibition compared to other hematopoietic cell lineages. Our results provide insight into ANKL genetics and a framework for application of targeted therapies in NK-cell malignancies.

The heart of a newborn mouse has an exceptional capacity to regenerate from myocardial injury that is lost within the first week of its life. In order to elucidate the molecular mechanisms taking place in the mouse heart during this critical period we applied an untargeted combinatory multiomics approach using large-scale mass spectrometry-based quantitative proteomics, metabolomics and mRNA sequencing on hearts from 1-day-old and 7-day-old mice. As a result, we quantified 1.937 proteins (366 differentially expressed), 612 metabolites (263 differentially regulated) and revealed 2.586 differentially expressed gene loci (2.175 annotated genes). The analyses pinpointed the fructose-induced glycolysis-pathway to be markedly active in 1-day-old neonatal mice. Integrated analysis of the data convincingly demonstrated cardiac metabolic reprogramming from glycolysis to oxidative phosphorylation in 7-days old mice, with increases of key enzymes and metabolites in fatty acid transport (acylcarnitines) and β-oxidation. An upsurge in the formation of reactive oxygen species and an increase in oxidative stress markers, e.g., lipid peroxidation, altered sphingolipid and plasmalogen metabolism were also evident in 7-days mice. In vitro maintenance of physiological fetal hypoxic conditions retained the proliferative capacity of cardiomyocytes isolated from newborn mice hearts. In summary, we provide here a holistic, multiomics view toward early postnatal changes associated with loss of a tissue regenerative capacity in the neonatal mouse heart. These results may provide insight into mechanisms of human cardiac diseases associated with tissue regenerative incapacity at the molecular level, and offer a prospect to discovery of novel therapeutic targets.

After the landmark approval of T-VEC, oncolytic viruses are finding their way to the clinics. However, response rates have still room for improvement, and unfortunately there are currently no available markers to predict responses for oncolytic immunotherapy. Interleukin 8 (IL-8) production is upregulated in many cancers and it also connects to several pathways that have been shown to impair the efficacy of adenoviral immunotherapy. We studied the role of IL-8 in 103 cancer patients treated with oncolytic adenoviruses. We found high baseline serum IL-8 concentration to be independently associated with poor prognosis (p<0.001). Further, normal baseline IL-8 was associated with improved prognostic potential of calculation of the neutrophil-to-lymphocyte ratio (p<0.001). Interestingly, a decrease in IL-8 concentration after treatment with oncolytic adenovirus predicted better overall survival (p<0.001) and higher response rate, although this difference was not significant (p=0.066). We studied the combination of adenovirus and IL-8 neutralizing antibody ex vivo in single cell suspensions and in co-cultures of tumor-associated CD15+ neutrophils and CD3+ tumor-infiltrating lymphocytes derived from fresh patient tumor samples. These results indicate a role for IL-8 as a biomarker in oncolytic virotherapy, but additionally provide a rationale for targeting IL-8 to improve treatment efficacy. In conclusion, curtailing the activity of IL-8 systemically or locally in the tumor microenvironment could improve anti-tumor immune responses resulting in enhanced efficacy of adenoviral immunotherapy of cancer., Competing Interests: CONFLICTS OF INTEREST A.H. is employee and shareholder in TILT Biotherapeutics Ltd. and shareholder in Targovax ASA.