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The challenge of eradicating leukemia in patients with acute myelogenous leukemia (AML) after initial cytoreduction has motivated modern efforts to combine synergistic active modalities including immunotherapy. Recently, the ETCTN/CTEP 10026 study tested the combination of the DNA methyltransferase inhibitor decitabine together with the immune checkpoint inhibitor ipilimumab for AML/myelodysplastic syndrome (MDS) either after allogeneic hematopoietic stem cell transplantation (HSCT) or in the HSCT-naïve setting. Integrative transcriptome-based analysis of 304 961 individual marrow-infiltrating cells for 18 of 48 subjects treated on study revealed the strong association of response with a high baseline ratio of T to AML cells. Clinical responses were predominantly driven by decitabine-induced cytoreduction. Evidence of immune activation was only apparent after ipilimumab exposure, which altered CD4+ T-cell gene expression, in line with ongoing T-cell differentiation and increased frequency of marrow-infiltrating regulatory T cells. For post-HSCT samples, relapse could be attributed to insufficient clearing of malignant clones in progenitor cell populations. In contrast to AML/MDS bone marrow, the transcriptomes of leukemia cutis samples from patients with durable remission after ipilimumab monotherapy showed evidence of increased infiltration with antigen-experienced resident memory T cells and higher expression of CTLA-4 and FOXP3. Altogether, activity of combined decitabine and ipilimumab is impacted by cellular expression states within the microenvironmental niche of leukemic cells. The inadequate elimination of leukemic progenitors mandates urgent development of novel approaches for targeting these cell populations to generate long-lasting responses. This trial was registered at www.clinicaltrials.gov as #NCT02890329.

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Transformation to aggressive disease histologies generates formidable clinical challenges across cancers, but biological insights remain few. We modeled the genetic heterogeneity of chronic lymphocytic leukemia (CLL) through multiplexed in vivo CRISPR-Cas9 B-cell editing of recurrent CLL loss-of-function drivers in mice and recapitulated the process of transformation from indolent CLL into large cell lymphoma [i.e., Richter syndrome (RS)]. Evolutionary trajectories of 64 mice carrying diverse combinatorial gene assortments revealed coselection of mutations in Trp53, Mga, and Chd2 and the dual impact of clonal Mga/Chd2 mutations on E2F/MYC and interferon signaling dysregulation. Comparative human and murine RS analyses demonstrated tonic PI3K signaling as a key feature of transformed disease, with constitutive activation of the AKT and S6 kinases, downmodulation of the PTEN phosphatase, and convergent activation of MYC/PI3K transcriptional programs underlying enhanced sensitivity to MYC/mTOR/PI3K inhibition. This robust experimental system presents a unique framework to study lymphoid biology and therapy.Significance:Mouse models reflective of the genetic complexity and heterogeneity of human tumors remain few, including those able to recapitulate transformation to aggressive disease histologies. Herein, we model CLL transformation into RS through multiplexed in vivo gene editing, providing key insight into the pathophysiology and therapeutic vulnerabilities of transformed disease.This article is highlighted in the In This Issue feature, p. 101

Three new species of Vappolotes Zhao & Li, 2019 (Araneae: Agelenidae) are described from Hunan and Sichuan provinces, China: V. longshan sp. n. (♀♂), V. tianjiayu sp. n. (♀), and V. hei sp. n. (♀♂). The diagnostic characters of this genus are amended based on the newly obtained data. Photographs of habitus and copulatory organs, as well as their distributions, are provided for all new Vappolotes species.

Non-uniform dynamics of single CpGs among CLL progression

Transformation to aggressive disease histologies generates formidable clinical challenges across cancers, but biological insights remain few. We modeled the genetic heterogeneity of chronic lymphocytic leukemia (CLL) through multiplexed in vivo CRISPR-Cas9 B-cell editing of recurrent CLL loss-of-function drivers in mice and recapitulated the process of transformation from indolent CLL into large cell lymphoma [i.e., Richter syndrome (RS)]. Evolutionary trajectories of 64 mice carrying diverse combinatorial gene assortments revealed coselection of mutations in Trp53, Mga, and Chd2 and the dual impact of clonal Mga/Chd2 mutations on E2F/MYC and interferon signaling dysregulation. Comparative human and murine RS analyses demonstrated tonic PI3K signaling as a key feature of transformed disease, with constitutive activation of the AKT and S6 kinases, downmodulation of the PTEN phosphatase, and convergent activation of MYC/PI3K transcriptional programs underlying enhanced sensitivity to MYC/mTOR/PI3K inhibition. This robust experimental system presents a unique framework to study lymphoid biology and therapy.Significance:Mouse models reflective of the genetic complexity and heterogeneity of human tumors remain few, including those able to recapitulate transformation to aggressive disease histologies. Herein, we model CLL transformation into RS through multiplexed in vivo gene editing, providing key insight into the pathophysiology and therapeutic vulnerabilities of transformed disease.This article is highlighted in the In This Issue feature, p. 101

Supplementary file contains a PDF version of supplementary figures S1-S7 and associated figure legends.

Table of correlation between time points within one sample and number of hyper- or hypomethylated CpGs for all CLL (top) and naïve and memory B cell samples (bottom)

Supplementary file contains all supplementary tables S1-S16 in Excel format in the order in which they appear in the text.

Supplementary Figures 1-7 and matching legends

Most human cancers converge to a deregulated methylome with reduced global levels and elevated methylation at select CpG islands. To investigate the emergence and dynamics of the cancer methylome, we characterized genome-wide DNA methylation in preneoplastic monoclonal B-cell lymphocytosis (MBL) and chronic lymphocytic leukemia (CLL), including serial samples collected across disease course. We detected the aberrant tumor-associated methylation landscape at CLL diagnosis and found no significant differentially methylated regions in the high-count MBL-to-CLL transition. Patient methylomes showed remarkable stability with natural disease and posttherapy progression. Single CLL cells were consistently aberrantly methylated, indicating a homogeneous transition to the altered epigenetic state and a distinct expression profile together with MBL cells compared with normal B cells. Our longitudinal analysis reveals the cancer methylome to emerge early, which may provide a platform for subsequent genetically driven growth dynamics, and, together with its persistent presence, suggests a central role in disease onset.Significance:DNA methylation data from a large cohort of patients with MBL and CLL show that epigenetic transformation emerges early and persists throughout disease stages with limited subsequent changes. Our results indicate an early role for this aberrant landscape in the normal-to-preneoplastic transition that may reflect a pan-cancer mechanism.See related commentary by Rossi, p. 6.This article is highlighted in the In This Issue feature, p. 1

Supplementary methods, Table S1, supplementary figures 1-9

While cancers evolve during disease progression and in response to therapy, temporal dynamics remain difficult to study in humans due to the lack of consistent barcodes marking individual clones in vivo. We employ mitochondrial single-cell assay for transposase-accessible chromatin with sequencing to profile 163,279 cells from 9 patients with chronic lymphocytic leukemia (CLL) collected across disease course and utilize mitochondrial DNA (mtDNA) mutations as natural genetic markers of cancer clones. We observe stable propagation of mtDNA mutations over years in the absence of strong selective pressure, indicating clonal persistence, but dramatic changes following tight bottlenecks, including disease transformation and relapse posttherapy, paralleled by acquisition of copy-number variants and changes in chromatin accessibility and gene expression. Furthermore, we link CLL subclones to distinct chromatin states, providing insight into nongenetic sources of relapse. mtDNA mutations thus mirror disease history and provide naturally occurring genetic barcodes to enable patient-specific study of cancer subclonal dynamics.Significance:Single-cell multi-omic profiling of CLL reveals the utility of somatic mtDNA mutations as in vivo barcodes, which mark subclones that can evolve over time along with changes in accessible chromatin and gene expression profiles to capture dynamics of disease evolution.See related commentary by Hilton and Scott, p. 2965.This article is highlighted in the In This Issue feature, p. 2945

Supplementary Table 2 - %CLL cells marked by mtDNA mutations

Purpose: Although local tissue-based immune responses are critical for elucidating direct tumor–immune cell interactions, peripheral immune responses are increasingly recognized as occupying an important role in anticancer immunity. We evaluated serial blood samples from patients with advanced epithelial ovarian cancer (EOC) undergoing standard-of-care neoadjuvant carboplatin and paclitaxel chemotherapy (including dexamethasone for prophylaxis of paclitaxel-associated hypersensitivity reactions) to characterize the evolution of the peripheral immune cell function and composition across the course of therapy. Experimental Design: Serial blood samples from 10 patients with advanced high-grade serous ovarian cancer treated with neoadjuvant chemotherapy (NACT) were collected before the initiation of chemotherapy, after the third and sixth cycles, and approximately 2 months after completion of chemotherapy. T-cell function was evaluated using ex vivo IFNγ ELISpot assays, and the dynamics of T-cell repertoire and immune cell composition were assessed using bulk and single-cell RNA sequencing (RNAseq). Results: T cells exhibited an improved response to viral antigens after NACT, which paralleled the decrease in CA125 levels. Single-cell analysis revealed increased numbers of memory T-cell receptor (TCR) clonotypes and increased central memory CD8+ and regulatory T cells throughout chemotherapy. Finally, administration of NACT was associated with increased monocyte frequency and expression of HLA class II and antigen presentation genes; single-cell RNAseq analyses showed that although driven largely by classical monocytes, increased class II gene expression was a feature observed across monocyte subpopulations after chemotherapy. Conclusions: NACT may alleviate tumor-associated immunosuppression by reducing tumor burden and may enhance antigen processing and presentation. These findings have implications for the successful combinatorial applications of immune checkpoint blockade and therapeutic vaccine approaches in EOC.

Purpose:Although local tissue-based immune responses are critical for elucidating direct tumor–immune cell interactions, peripheral immune responses are increasingly recognized as occupying an important role in anticancer immunity. We evaluated serial blood samples from patients with advanced epithelial ovarian cancer (EOC) undergoing standard-of-care neoadjuvant carboplatin and paclitaxel chemotherapy (including dexamethasone for prophylaxis of paclitaxel-associated hypersensitivity reactions) to characterize the evolution of the peripheral immune cell function and composition across the course of therapy.Experimental Design:Serial blood samples from 10 patients with advanced high-grade serous ovarian cancer treated with neoadjuvant chemotherapy (NACT) were collected before the initiation of chemotherapy, after the third and sixth cycles, and approximately 2 months after completion of chemotherapy. T-cell function was evaluated using ex vivo IFNγ ELISpot assays, and the dynamics of T-cell repertoire and immune cell composition were assessed using bulk and single-cell RNA sequencing (RNAseq).Results:T cells exhibited an improved response to viral antigens after NACT, which paralleled the decrease in CA125 levels. Single-cell analysis revealed increased numbers of memory T-cell receptor (TCR) clonotypes and increased central memory CD8+ and regulatory T cells throughout chemotherapy. Finally, administration of NACT was associated with increased monocyte frequency and expression of HLA class II and antigen presentation genes; single-cell RNAseq analyses showed that although driven largely by classical monocytes, increased class II gene expression was a feature observed across monocyte subpopulations after chemotherapy.Conclusions:NACT may alleviate tumor-associated immunosuppression by reducing tumor burden and may enhance antigen processing and presentation. These findings have implications for the successful combinatorial applications of immune checkpoint blockade and therapeutic vaccine approaches in EOC.

Supplementary Data from Mitochondrial DNA Mutations as Natural Barcodes for Lineage Tracing of Murine Tumor Models

Murine models are indispensable tools for functional genomic studies and preclinical testing of novel therapeutic approaches. Mitochondrial single-cell assay for transposase-accessible chromatin using sequencing (mtscATAC-seq) enables the dissection of cellular heterogeneity and clonal dynamics by capturing chromatin accessibility, copy-number variations (CNV), and mitochondrial DNA (mtDNA) mutations, yet its applicability to murine studies remains unexplored. By leveraging mtscATAC-seq in novel chronic lymphocytic leukemia and Richter syndrome mouse models, we report the detection of mtDNA mutations, particularly in highly proliferative murine cells, alongside CNV and chromatin state changes indicative of clonal evolution upon secondary transplant. This study thus demonstrates the feasibility and utility of multi-modal single-cell and natural barcoding approaches to characterize murine cancer models.Significance:mtDNA mutations can serve as natural barcodes to enable lineage tracing in murine cancer models, which can be used to provide new insights into disease biology and to identify therapeutic vulnerabilities.

Supplementary Figure from Improved T-cell Immunity Following Neoadjuvant Chemotherapy in Ovarian Cancer

Supplementary Table from Improved T-cell Immunity Following Neoadjuvant Chemotherapy in Ovarian Cancer

Supplementary Data from Improved T-cell Immunity Following Neoadjuvant Chemotherapy in Ovarian Cancer

Great challenges still remain in the management of patients with castration-resistant prostate cancer (CRPC) based on traditional treatments, and the rapid development of nanotechnology may find a breakthrough. Herein, a novel type of multifunctional self-assembly magnetic nanocarriers (IR780-MNCs) containing iron oxide nanoparticles (Fe3O4 NPs) and IR780 iodide was synthesized by an optimized process. With a hydrodynamic diameter of 122 nm, a surface charge of –28.5 mV and the drug loading efficiency of 89.6%, IR780-MNCs have increased cellular uptake efficiency, long-term stability, ideal photothermal conversion ability and excellent superparamagnetic behavior. The in vitro study indicated that IR780-MNCs have excellent biocompatibility and could induce significant cell apoptosis under the 808 nm laser irradiation. The in vivo study showed that IR780-MNCs highly accumulated at the tumor area could reduce the tumor volume of tumor-bearing mice by 88.5% under the 808 nm laser irradiation, but minimal damage to surrounding normal tissues. Since IR780-MNCs encapsulated a large number of 10 nm homogeneous spherical Fe3O4 NPs, which can be used as T2 contrast agent, the best window for photothermal therapy can be determined through MRI. In conclusion, IR780-MNCs have initially showed excellent antitumor effect and biosafety in the treatment of CRPC. This work provides novel insights into the precise treatment of CRPC by using a safe nanoplatform based on the multifunctional nanocarriers.

Baiyuerius Li, Zhao & Li gen. n., a new genus of the subfamily Coelotinae F. O. Pickard-Cambridge, 1893 is described, including five new species: B. daxi Li, Zhao & Li sp. n. (♀), B. pindong Li, Zhao & Li sp. n. (♂), B. tamdao Li, Zhao & Li sp. n. (♀), B. zhuping Li, Zhao & Li sp. n. (♂), and B. zuojiang Li, Zhao & Li sp. n. (♂♀), from southern China and the north of Vietnam. Our molecular analysis supports Baiyuerius Li, Zhao & Li gen. n. as monophyletic and as a sister group of the newly established genus Yunguirius.

Fracture is one of the most common traumatic diseases in clinical practice, and metal plates have always been the first choice for fracture treatment because of their high strength. However, the bone plates have high elastic modulus and do not match the biomechanics of human bone, which adversely affects callus formation and fracture healing. Moreover, the complex microenvironment in the human body can induce corrosion of metallic materials and release toxic ions, which reduces the biocompatibility of the bone plate, and may necessitate surgical removal of the implant. In this study, tantalum (Ta) was deposited on porous silicon carbide (SiC) scaffolds by chemical vapor deposition technology to prepare a novel porous tantalum (pTa) trabecular bone metal plate. The function of the novel bone plate was evaluated by implantation in an animal fracture model. The results showed that the novel bone plate was effective in fracture fixation, without breakage. Both X-ray and microcomputed tomography analysis showed indirect healing by both pTa trabecular bone metal plates and titanium (Ti) plates; however, elastic fixation and obvious callus formation were observed after fixation with pTa trabecular bone metal plates, indicating better bone repair. Histology showed that pTa promoted the formation of new bone and integrated well with the host bone. Therefore, this novel pTa trabecular bone metal plate has good prospects for application in treating fractures.

Sea–land transition caused by marine incursion and regression is hypothesized to be a major driving force in terrestrial biodiversity, providing opportunities for marine ancestors to colonize terrestrial habitats and driving vicariant speciation in distinct geographical regions. Here, we test this hypothesis in East Asia using amphipods of the Morinoia japonica complex. We constructed a dataset from 269 individuals covering all known ranges of this species complex. Phylogenetic and biogeographic analyses revealed that sea–land transition during the Miocene drove the coastal ancestor to invade terrestrial habitats in East Asia and subsequently split into eight biogeographic lineages in eastern China, Korea, Japan and some Pacific islands. Stepping-stone dispersal resulted in a relatively wide distribution of M. japonica, and long-term geographical isolation led to the diversification of the M. japonica complex. Species delimitation analysis suggests that this complex contains eight species. We describe the geographical group from eastern China as a new species, Morinoia aosen sp. nov., based on genetic and morphological comparisons with other geographical groups. Type specimens are deposited in the Institute of Zoology, Chinese Academy of Sciences (IZCAS) in Beijing, China.

Cenozoic tectonic evolution in the Tethyan region has greatly changed the landforms and environment of Eurasia, driving the evolution of animals and greatly affecting the diversity patterns of Eurasian animals. By combining the latest Tethyan paleogeographic models and some recently published Eurasian zoological studies, we systematically summarize how tectonic evolution in the Tethyan region has influenced the evolution and diversity patterns of Eurasian animals. The convergence of continental plates, closure of Tethys Sea, and Tethyan sea-level changes have directly affected the composition and spatial distribution of Eurasian animal diversity. The topographic and environmental changes caused by Tethyan tectonics have determined regional animal diversity in Eurasia by influencing animal origin, dispersal, preservation, diversification, and extinction. The ecological transformations resulted in the emergence of new habitats and niches, which promoted animal adaptive evolution, specialization, speciation, and expansion. We highlight that the Cenozoic tectonic evolution of the Tethyan region has been responsible for much of the alteration in Eurasian animal distribution and has been an essential force in shaping organic evolution. Furthermore, we generalize a general pattern that Tethyan geological events are linked with Eurasian animal evolution and diversity dynamics.特提斯海区域新生代以来的构造演化极大地改变了欧亚地区的地貌和环境，推动了欧亚地区的动物演化，并深刻影响了欧亚地区的动物多样性格局。该文通过结合最新的特提斯海区域古地理模型，对近期发表的一系列欧亚地区动物学研究进行系统回顾和总结，详细论述了特提斯海区域新生代以来的构造演化是如何影响欧亚地区动物的演化及多样性格局的：大陆板块的汇聚、特提斯海的闭合以及特提斯海区域海平面变化直接影响了欧亚地区的动物多样性组成和空间分布；海陆变迁引起的区域地理和环境变化，对当地动物的起源、扩散、存留、多样化和灭绝产生了一系列影响，进而决定了欧亚动物的区域多样性格局；生态环境的变化导致了新栖息地和新生态位的出现，促进了欧亚动物的适应性进化、特化、物种形成和扩张。我们强调了特提斯海区域新生代构造演化是欧亚动物分布变化的主要原因，也是驱动欧亚动物演化的重要力量。此外，我们提出了特提斯地质事件与欧亚动物演化和多样性动态相关的一般模式。.

While cancers evolve during disease progression and in response to therapy, temporal dynamics remain difficult to study in humans due to the lack of consistent barcodes marking individual clones in vivo. We employ mitochondrial single-cell assay for transposase-accessible chromatin with sequencing to profile 163,279 cells from 9 patients with chronic lymphocytic leukemia (CLL) collected across disease course and utilize mitochondrial DNA (mtDNA) mutations as natural genetic markers of cancer clones. We observe stable propagation of mtDNA mutations over years in the absence of strong selective pressure, indicating clonal persistence, but dramatic changes following tight bottlenecks, including disease transformation and relapse posttherapy, paralleled by acquisition of copy-number variants and changes in chromatin accessibility and gene expression. Furthermore, we link CLL subclones to distinct chromatin states, providing insight into nongenetic sources of relapse. mtDNA mutations thus mirror disease history and provide naturally occurring genetic barcodes to enable patient-specific study of cancer subclonal dynamics.Significance:Single-cell multi-omic profiling of CLL reveals the utility of somatic mtDNA mutations as in vivo barcodes, which mark subclones that can evolve over time along with changes in accessible chromatin and gene expression profiles to capture dynamics of disease evolution.See related commentary by Hilton and Scott, p. 2965.This article is highlighted in the In This Issue feature, p. 2945

Fourteen symphytognathid species belonging to three genera are examined, including the descriptions of eight new species and two new genera from China, Vietnam, Thailand and Myanmar: Patu Marples, 1951: P. catba S. Li & Lin, sp. nov. (♂, Vietnam), P. dakou S. Li & Lin, sp. nov. (♂♀, China), P. damtao S. Li & Lin, sp. nov. (♂, Vietnam), P. jiangzhou S. Li & Lin, sp. nov. (♀, China), P. jidanweishi Miller, Griswold & Yin, 2009 (♂♀, China), P. nagarat S. Li & Lin, sp. nov. (♂♀, Thailand), P. nigeri Lin & S. Li, 2009 (♀, China), P. putao S. Li & Lin, sp. nov. (♀, Myanmar), P. qiqi Miller, Griswold & Yin, 2009 (♀, China) and P. xiaoxiao Miller, Griswold & Yin, 2009 (♂♀, China); Kirinua S. Li & Lin, gen. nov.: K. maguai S. Li & Lin, sp. nov. (♂♀, China) and K. yangshuo S. Li & Lin, sp. nov. (♂♀, China); Swilda S. Li & Lin, gen. nov.: S. longtou (Miller, Griswold & Yin, 2009), comb. nov. (♂♀, China) is transferred from Crassignatha Wunderlich, 1995 and S. spinathoraxi (Lin & S. Li, 2009), comb. nov. (♂♀, China) is transferred from Patu. Diagnoses, descriptions and illustrations are provided for new taxa, as well as a distribution map. The males of P. xiaoxiao and S. longtou are described for the first time. Type specimens of P. jidanweishi, P. nigeri, P. qiqi, P. xiaoxiao, S. longtou and S. spinathoraxi are re-examined and photographed. All Asian Patu species are revised and two species, P. kishidai Shinkai, 2009 and P. bispina Lin, Pham & S. Li, 2009, are transferred to Crassignatha and proposed as new combinations: Crassignatha kishidaicomb. nov. and C. bispinacomb. nov. In addition, DNA barcodes and genetic distances of ten species treated in this paper were obtained to confirm identification.

Volcano-tectonic processes have been viewed as primary drivers in the formation of present-day diversity. Volcanos associated with mountain uplifts drive allopatric speciation through vicariance and may impact the surrounding areas like species pump or species attractor. However, the application of these hypotheses to aquatic fauna has rarely been tested explicitly. We tested these hypotheses in the Changbai Mountains (Mts), which are one of the most typical, active volcanic ranges in Northeast (NE) Asia with a long and turbulent geological history. The Gammarus nekkensis species complex of amphipod crustaceans, widely distributed throughout NE Asia with poor dispersal abilities and a long evolutionary history, is a suitable model for testing hypotheses of species pump or species attractor. Phylogenetic and ancestral range reconstructions demonstrated that the studied amphipod originated from the Changbai Mts ~27 Ma and diverged into eastern (Clade I) and western (Clade II) clades, which corresponds well with the initial volcanic eruption of the Changbai Mts in the Late Oligocene. The subsequent diversifications of subclades CI-3, CII-1a and CII-2a were probably driven by second and third eruptions of the Changbai Mts during the Miocene. In particular, the Changbai lineages had spread to the Russian Far East multiple times since the Early Miocene, and widely colonized the region during the Pleistocene. Our discoveries suggest that the ancient volcanos of the Changbai Mts act as species pumps in NE Asia, resulted in burst of diversification around the Changbai Mts and subsequent dispersals into adjacent regions.