A functional SUMO-interacting motif in the transactivation domain of c-Myb regulates its myeloid transforming ability

c-Myb is an essential hematopoietic transcription factor that controls proliferation and differentiation of progenitors during blood cell development. Whereas sumoylation of the C-terminal regulatory domain (CRD) is known to have a major impact on the activity of c-Myb, no role for noncovalent binding of small ubiquitin-like modifier (SUMO) to c-Myb has been described. Based on the consensus SUMO-interacting motif (SIM), we identified and examined putative SIMs in human c-Myb. Interaction and reporter assays showed that the SIM in the in the transactivation domain of c-Myb (V 267 NIV) is functional. This motif is necessary for c-Myb to be able to interact noncovalently with SUMO, preferentially SUMO2/3. Destroying the SUMO-binding properties by mutation resulted in a large increase in the transactivation potential of c-Myb. Mutational analysis and overexpression of conjugation-defective SUMO argued against intramolecular repression caused by sumoylated CRD and in favor of SUMO-dependent repression in trans. Using both a myeloid cell line-based assay and a primary hematopoietic cell assay, we addressed the transforming abilities of SUMO binding and conjugation mutants. Interestingly, only loss of SUMO binding, and not SUMO conjugation, enhanced the myeloid transformational potential of c-Myb. c-Myb with the SIM mutated conferred a higher proliferative ability than the wild-type and caused an effective differentiation block. This establishes SUMO binding as a mechanism involved in modulating the transactivation activity of c-Myb, and responsible for keeping the transforming potential of the oncoprotein in check

Abstract 2116: VPS34 inhibitor SB02024 activates cGAS-STING signaling and sensitizes tumors to STING agonist

Abstract Novel approaches to reduce tumor immunosuppression and improve responses to anti-cancer immunotherapies based on immune-checkpoint inhibitors are needed. Emerging evidence demonstrates that autophagy inhibition enhances anti-tumor immunity by tumor cell-intrinsic and extrinsic mechanisms. Recently, we reported that pharmacological inhibition of VPS34 (PIK3C3), a lipid kinase regulating autophagy initiation, decreases tumor growth and improve the efficacy of anti-PD-1/PD-L1 therapy in melanoma and colorectal cancer mouse models. This effect was dependent on increasing T and NK cell infiltration as well as the expression of CCL5 and CXCL10 chemokines in the tumor microenvironment. Here, we explored the signaling mechanisms underlying the chemokine release following treatment with VPS34 inhibitor SB02024. We found that both pharmacological and RNAi-mediated inhibition of VPS34 activated a cGAS-STING-dependent type I interferon response in renal cell carcinoma (RCC) and melanoma cell lines. Furthermore, combination treatment of VPS34 inhibitor SB02024 with STING agonist ADU-S100 or cGAMP increased the mRNA expression and release of proinflammatory cytokines in human and murine RCC and melanoma cancer cell lines. Oral administration of SB02024 in combination with intratumoral injections of ADU-S100 significantly decreased tumor growth and weight and improved mice survival of B16-F10 tumor-bearing mice. Taken together, our data demonstrates that targeting of VPS34 results in a cGAS/STING-mediated increase of pro-inflammatory cytokine secretion and synergizes with a STING agonist. We believe that systemic VPS34 inhibition using SB02024 would be of major interest in combination or as an alternative to STING agonists to improve anti-tumor immune responses. Citation Format: Yasmin Yu, Muhammad Z. Noman, Santiago Parpal, Simone C. Kleinendorst, Kristine B. Saether, Andrey Alexeyenko, Jenny Viklund, Martin Andersson, Jessica Martinsson, Katja P. Tamm, Angelo De Milito, Bassam Janji. VPS34 inhibitor SB02024 activates cGAS-STING signaling and sensitizes tumors to STING agonist [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2116

Recommendations for Bioinformatics in Clinical Practice

Abstract Next Generation Sequencing (NGS) is increasingly used in clinical diagnostics, largely driven by the success and robustness of Whole Genome Sequencing (WGS). Whereas updated guidelines exist for how to interpret and report on variants that are identified from NGS using bioinformatics pipelines, there is a need for standardised bioinformatics practices for diagnostics to ensure clinical consensus, accuracy, reproducibility and comparability of the results. This article presents consensus recommendations developed by 13 clinical bioinformatics units taking part in the Nordic Alliance for Clinical Genomics (NACG), by expert bioinformaticians working in clinical production. The recommendations are based on clinical practice and focus on analysis types, test and validation, standardisation and accreditation, as well as core competencies and technical management required for clinical bioinformatics operations. Key recommendations include adopting the hg38 genome build as the reference and a standard set of recommended analyses, including the use of multiple tools for structural variant (SV) calling and in-house data sets for filtering recurrent calls. Clinical bioinformatics production should operate under the ISO 15189 standard, utilising off-grid clinical-grade high-performance computing systems, standardised file formats, and strict code version control. Containerized software containers or environment management systems are needed to ensure reproducibility. Pipelines should be rigorously documented and tested for accuracy and reproducibility, minimally covering unit, integration, and end-to-end testing. Standard truth sets such as GIAB and SEQC2 for germline and somatic variant calling, respectively, should be supplemented by recall testing of previously validated clinical cases. Data integrity must be verified using file hashing, and sample identity should be checked via sample fingerprinting and genetically inferred identification markers such as sex and relatedness. Finally, clinical bioinformatics teams should encompass diverse skills, including software development, data management, quality assurance, and domain expertise in human genetics. These recommendations provide a consensus framework for standardising bioinformatics practices across clinical WGS applications and can serve as a practical guide to facilities that are new to large-scale sequencing-based diagnostics, or as a reference for those who already run high-volume clinical production using NGS

Spatiotemporal variation in reproductive parameters of yellow-bellied marmots

Spatiotemporal variation in reproductive rates is a common phenomenon in many wildlife populations, but the population dynamic consequences of spatial and temporal variability in different components of reproduction remain poorly understood. We used 43 years (1962-2004) of data from 17 locations and a capture-mark-recapture (CMR) modeling framework to investigate the spatiotemporal variation in reproductive parameters of yellow-bellied marmots (Marmota flaviventris), and its influence on the realized population growth rate. Specifically, we estimated and modeled breeding probabilities of two-year-old females (earliest age of first reproduction), > 2-year-old females that have not reproduced before (subadults), and > 2-year-old females that have reproduced before (adults), as well as the litter sizes of two-year old and > 2-year-old females. Most reproductive parameters exhibited spatial and/or temporal variation. However, reproductive parameters differed with respect to their relative influence on the realized population growth rate (lambda). Litter size had a stronger influence than did breeding probabilities on both spatial and temporal variations in lambda. Our analysis indicated that lambda was proportionately more sensitive to survival than recruitment. However, the annual fluctuation in litter size, abetted by the breeding probabilities, accounted for most of the temporal variation in lambda