Single-nucleus multiomic mapping of m6A methylomes and transcriptomes in native populations of cells with sn-m6A-CT.

N6-methyladenosine (m6A) RNA modification plays important roles in the governance of gene expression and is temporally regulated in different cell states. In contrast to global m6A profiling in bulk sequencing, single-cell technologies for analyzing m6A heterogeneity are not extensively established. Here, we developed single-nucleus m6A-CUT&Tag (sn-m6A-CT) for simultaneous profiling of m6A methylomes and transcriptomes within a single nucleus using mouse embryonic stem cells (mESCs). m6A-CT is capable of enriching m6A-marked RNA molecules in situ, without isolating RNAs from cells. We adapted m6A-CT to the droplet-based single-cell omics platform and demonstrated high-throughput performance in analyzing nuclei isolated from thousands of cells from various cell types. We show that sn-m6A-CT profiling is sufficient to determine cell identity and allows the generation of cell-type-specific m6A methylome landscapes from heterogeneous populations. These indicate that sn-m6A-CT provides additional dimensions to multimodal datasets and insights into epitranscriptomic landscape in defining cell fate identity and states.

BRAF(V600E) mutation together with loss of Trp53 or pTEN drives the origination of hairy cell leukemia from B-lymphocytes.

Hairy cell leukemia (HCL) is a B-lymphoma induced by BRAF(V600E) mutation. However, introducing BRAF(V600E) in B-lymphocytes fails to induce hematological malignancy, suggesting that BRAF(V600E) needs concurrent mutations to drive HCL ontogeny. To resolve this issue, here we surveyed human HCL genomic sequencing data. Together with previous reports, we speculated that the tumor suppressor TP53, P27, or PTEN restrict the oncogenicity of BRAF(V600E) in B-lymphocytes, and therefore that their loss-of-function facilitates BRAF(V600E)-driven HCL ontogeny. Using genetically modified mouse models, we demonstrate that indeed BRAF(V600E)KI together with Trp53KO or pTENKO in B-lymphocytes induces chronic lymphoma with pathological features of human HCL. To further understand the cellular programs essential for HCL ontogeny, we profiled the gene expression of leukemic cells isolated from BRAF(V600E)KI and Trp53KO or pTENKO mice, and found that they had similar but different gene expression signatures that resemble that of M2 or M1 macrophages. In addition, we examined the expression signature of transcription factors/regulators required for germinal center reaction and memory B cell versus plasma cell differentiation in these leukemic cells and found that most transcription factors/regulators essential for these programs were severely inhibited, illustrating why hairy cells are arrested at a transitional stage between activated B cells and memory B cells. Together, our study has uncovered concurrent mutations required for HCL ontogeny, revealed the B cell origin of hairy cells and investigated the molecular basis underlying the unique pathological features of the disease, with important implications for HCL research and treatment.

A PDZ Protein GIPC3 Positively Modulates Hedgehog Signaling and Melanoma Growth.

The hedgehog (Hh) pathway is essential for animal development, but aberrant activation promotes cancer growth. In this study, we show that GIPC3, a PDZ domain-containing protein with putative adaptor protein function, positively modulates Hh target gene expression in normal fibroblasts and melanoma cells and supports melanoma tumor growth. Using overexpression and epistasis studies, we show that Gipc3 potentiates Hh transcriptional output and that it modulates GLI-dependent transcription independently of Sufu. Whereas we find that GIPC3 protein does not interact with Hh pathway components, Ingenuity Pathway Analyses of GIPC3-interacting proteins identified by coimmunoprecipitation and mass spectrometry show an association with cancer pathogenesis. Subsequent interrogation of The Cancer Genome Atlas and the Human Protein Atlas databases reveals GIPC3 upregulation in many cancers. Using expression screens in selected groups of GIPC3-upregulated cancers with reported Hh pathway activation, we find a significant positive correlation of GIPC3 expression with Hh pathway components GLI1, GLI2, and GPR161 in melanoma lines. Consistently, GIPC3 knockdown in melanoma lines significantly reduces GLI1 and GLI2 expression, cell viability, colony formation, and allograft tumor growth. Our findings highlight previously unidentified roles of GIPC3 in potentiating Hh response and melanoma tumorigenesis and suggest that GIPC3 modulation on Hh signaling may be targeted to reduce melanoma growth.

Impact of a SBRT/SRS longitudinal telehealth training pilot course in Latin America.

PURPOSE: To assess the impact of longitudinal telehealth training in stereotactic body radiation therapy (SBRT) and stereotactic radiosurgery (SRS) for clinicians in Latin America. MATERIALS AND METHODS: Professionals from two Peruvian centers received an initial SBRT/SRS on-site training course and subsequently received follow-up telehealth training (interventional group) or not (negative control arm). Twelve live video conference sessions were scheduled. Surveys pre- and post-curriculum measured participants' confidence in seven practical domains of SBRT/SRS, based on Likert scales of 1-5, and post-curriculum surveys assessed educators' experiences. RESULTS: Sixty-one participants were registered, with an average of 24 attendees per session. Pre- and post- surveys were completed by 22 participants. For interventional and negative-control groups, mean changes in Likert scale were satisfactory for the former and remained unmodified for the latter. CONCLUSIONS: Conducting telehealth educational programs via virtual classroom sessions could be a reliable method to augment training for SBRT and SRS.