Citations

Abstract Forward genetic screens are powerful tools for novel biological discoveries because they do not require a priori knowledge of genes controlling a phenotype of interest. The Sleeping Beauty (SB) transposon system is one of many commonly employed genetic screen tools used to induce both gain and loss of gene function mutations via insertional mutagenesis. In the past, SB screens have been successfully utilized to discover several cancer driver genes that could serve as potential therapeutic targets. More recently, SB technologies have been extended to other applications, including the discovery of mechanisms contributing to drug resistance and novel targets for immunotherapy. Importantly, existing tools for SB screen data analysis do not support data-driven case–control comparisons capable of analyzing screens with this design. To this end, we developed a network-based common insertion site (NetCIS) analysis tool to robustly identify common insertion sites (CISes) in a case–control phenotype selection screen. NetCIS uses an efficient graph-based algorithm for discovering statistically significant CISes that differ between cases and controls. We benchmark NetCIS against other insertional mutagenesis analysis tools using a previously published SB dataset and show that NetCIS is able to prioritize previous biologically validated genes similarly or better than competing methods, is the only method that effectively utilizes a case–control experimental setup, and can identify CISes involving unannotated regions of the genome that are overlooked by existing methods. Code for NetCIS and an accompanying tutorial can be found at https://github.com/RogersLabGroup/NetCIS.

Abstract Medulloblastoma (MB) is one of the most prevalent malignant brain tumors in children, with tremendous cognitive and neuroendocrine disability among survivors. Group 3 (G3) MBs have poor overall survival at < 50%, higher frequency of metastasis, and no targeted therapies. Amplification of MYC and activation of TGFβ signaling are frequent in G3MB. Remarkably, some tumors have no reported mutations, suggesting roles for epigenetic mechanisms in driving disease. We have generated novel humanized models for G3MB by transducing neuroepithelial stem cells (NESC) derived from human induced pluripotent stem (iPSC) cells with TGFβ effectors alone and/or in combination with MYC, prioritizing combinations observed in patients. We determined that both MYC and TGFβ effectors drove tumor formation in vivo with the combination driving more aggressive tumors and higher frequency of metastasis. NESC-derived tumors clustered with human G3/G4MB, indicating our models recapitulated human disease. To decipher mechanisms of resistance, we integrated CUT&RUN for MYC genomic localization and histone PTMs with RNA-sequencing and discovered MYC-bound neural developmental genes repressed in MYC and TGFβR1-driven lines. Further interrogation identified JARID1B, a MYC binding partner, as a regulator of many of these repressed neural genes. We postulate that deregulation of MYC and context-specific targeting to the chromatin template culminates in transcriptional alterations that promote aggressive G3MB. To further investigating therapeutic vulnerabilities, we have performed a in vivo whole genome CRISPRi screen to reveal important drivers of malignancy, one of the first of it’s kind in brain tumors. We are functionally validating key candidate sgRNAs that were differentially significant in primary tumors and may also have key roles in driving metastasis. Excitingly, many sgRNAs identified from the screen were chromatin modifiers and may be important co-factors of MYC-mediated regulation on the epigenome. Collectively, our studies provide a critical framework for understanding mechanisms of resistance and the epigenetic landscape of MYC- and TGFβ-driven MB.

ABSTRACT Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas with poor prognosis and a strong tendency for metastasis and relapse. Surgical removal remains the mainstay of treatment but is frequently ineffective or impractical. Currently, no effective targeted therapy exists for this type of malignancy. PRMT5 has recently emerged as a promising therapeutic target in various human cancers with MTAP loss, which results in cancer cell dependency on PRMT5 activity. The frequent loss of MTAP in MPNSTs suggests that PRMT5 inhibition is a promising therapeutic option and enables the stratification of cancer patients with few treatment options. We first examined human nerve sheath tumor samples and found that increased PRMT5 expression and activity correlated with MTAP loss in 86.8% (33/38) of MPNSTs and in atypical neurofibromatous neoplasm with uncertain biologic potential (ANNUBP) (5/5). When PRMT5 activity was inhibited genetically and chemically, the cell growth of MTAP-deficient MPNST cell lines was suppressed, but not that of MTAP-proficient MPNST cell lines. Moreover, in the PRMT5-inhibited MTAP-deficient MPNST cell lines, spontaneous DNA damage accumulation was observed following G2/M cell cycle arrest. The DNA replication stress marker RPA32 decreased, and CHK1 was activated early after PRMT5 knockdown, likely contributing to the accumulation of DNA damage. In addition, we combined PRMT5 inhibition with the DNA-damaging agents doxorubicin and gemcitabine, resulting in synergistic effects and increased cancer cell death in MTAP-deficient MPNST cell lines. Together, these findings identify PRMT5 as a compelling therapeutic target in MTAP-deficient MPNSTs. This PRMT5 inhibition strategy has strong translational potential for MPNSTs. GRAPHICAL ABSTRACT

Abstract Motivation Intratumor heterogeneity arises from ongoing somatic evolution and complicates cancer diagnosis, prognosis, and treatment. Reconstructing evolutionary dynamics typically requires spatiotemporal samples, which are often unavailable in clinical settings. Computational approaches that can infer tumor evolutionary history from single-timepoint bulk sequencing data remain limited. Results We present estimating evolutionary events through single-timepoint sequencing (TEATIME), a novel computational framework that models tumors as mixtures of two competing cell populations: an ancestral clone with baseline fitness and a derived subclone with elevated fitness. Using cross-sectional bulk sequencing data, TEATIME estimates mutation rates, timing of subclone emergence, relative fitness, and number of generations of growth. To quantify intratumor fitness asymmetries, we introduce a novel metric—fitness diversity—which captures the imbalance between competing cell populations and serves as a measure of functional intratumor heterogeneity. Applying TEATIME to 33 tumor types from The Cancer Genome Atlas, we revealed divergent as well as convergent evolutionary patterns. Notably, we found that immune-hot microenvironments constraint subclonal expansion and limit fitness diversity. Moreover, we detected temporal dependencies in mutation acquisition, where early driver mutations in ancestral clones epistatically shape the fitness landscape, predisposing specific subclones to selective advantages. These findings underscore the importance of intratumor competition and tumor-microenvironment interactions in shaping evolutionary trajectories, driving intratumor heterogeneity. Lastly, we demonstrate that TEATIME-derived evolutionary parameters and fitness diversity offer novel prognostic insights across multiple cancer types. Availability and implementation R implementation of TEATIME is available on GitHub (https://github.com/liliulab/TEATIME) and Zenodo (https://zenodo.org/records/17422174).

Abstract Platinum-based chemotherapy remains a cornerstone of treatment for triple-negative breast cancer (TNBC), yet the molecular determinants governing platinum response remain poorly defined. By leveraging the randomized Phase II INFORM trial, which compared neoadjuvant cisplatin to anthracycline-based therapy in BRCA1/2 -mutant breast cancer—we identified miR-362-3p as a specific regulator of cisplatin sensitivity. Higher plasma miR-362-3p expression was exclusively associated with favorable clinical outcome in the cisplatin arm, with no association observed in the AC arm, decoupling platinum-specific vulnerability from general chemotherapy response. We used gain- and loss-of-function TNBC models to establish that miR-362-3p functions as a potent sensitizer to cisplatin in vitro and in vivo . Integrated TCGA analysis and experimental validation identified BCLAF1, a key regulator of DNA damage response, as a direct repression target of miR-362-3p. We uncovered a novel role for the miR-362-3p/ BCLAF1 axis in overcoming platinum resistance in TNBC.

Summary Family history of haematological malignancy (FHHM) reflects inherited susceptibility to non‐Hodgkin lymphoma (NHL), but its prognostic significance remains poorly understood. We used a cohort study of 1994 NHL patients to evaluate associations between FHHM and clinical outcomes across lymphoma subtypes. We estimated associations, adjusted for sex and lymphoma‐specific prognostic index, of self‐reported FHHM in a first‐degree relative with overall survival (OS), event‐free survival (EFS) and lymphoma‐specific survival (LSS) using multivariable Cox regression models and with failure to achieve EFS at 24 months (EFS24) using logistic regression. FHHM prevalence was 12.8%, and the median follow‐up among survivors was 12.0 years. FHHM was not associated with OS. In subtype‐specific analyses, FHHM was associated with inferior LSS (hazard ratio [HR] = 1.98, 95% confidence interval [CI] 1.11–3.53) in follicular lymphoma (FL); this association and the association with EFS24 failure strengthened among those treated with front‐line immunochemotherapy (EFS24 failure odds ratio = 2.57, 95% CI 1.08–5.99; LSS HR = 2.68, 95% CI 1.09–6.58). In mantle cell lymphoma, FHHM was associated with inferior EFS (HR = 1.70, 95% CI 1.02–2.85). Interaction testing did not demonstrate significant heterogeneity of FHHM effects by lymphoma subtype. FHHM may be associated with adverse outcomes in select NHL groups, particularly FL. These findings warrant further investigation of germline and environmental factors potentially contributing to more aggressive FLs.

CTNNB1- mutated hepatocellular carcinomas are characterized by a distinctive morphology and activation of the Wnt pathway. AXIN1 also plays a key role in the Wnt pathway, but the morphology of AXIN1-mutated tumors has not been examined. In addition, there are ongoing questions on the ability of AXIN1 mutations to activate the Wnt pathway in hepatocellular carcinoma. AXIN1 mutated tumors (N=18) were studied, along with control groups: CTNNB1 (N=17), APC (6), or “Other” genes in the Wnt pathway (5). Wnt pathway activation was studied by immunostains for beta-catenin and glutamine synthetase. Findings were supplemented by gene expression analysis using TCGA data. On histologic examination, the classic morphology associated with beta-catenin mutations was found in all 4 groups: 8/18 AXIN1 (44%), 10/17 CTNNB1 (59%), 4/6 APC (67%), and 1/5 Other (20%). By immunohistochemistry, Wnt pathway activation was found in 11/18 AXIN1 (61%), 15/17 CTTNB1 (88%), 6/6 APC (100%), and 5/5 (100%) of Other. In AXIN1- mutated tumors, the Wnt pathway was weakly activated. Glutamine synthetase stains also highlighted a new “progressed pattern” associated with distinct subnodules of staining. Tertiary lymphoid structures were uncommon except for cases with CTTNNB1 mutations plus additional mutations in the Wnt pathway. In summary, the classic morphology associated with CTNNB1 mutations is found in hepatocellular carcinomas with mutations in AXIN1 , APC , and other Wnt genes. AXIN1 mutated tumors have Wnt activation that is detectable but at lower levels than CTNNB1 mutated tumors. As tumors progress, their level of Wnt activation can change.

ABSTRACT 162 patients with relapsed secondary central nervous system lymphoma (R‐SCNSL) (median age, 65 years; male, 59.9%) including central nervous system (CNS)‐only ( n = 120) and concomitant CNS/systemic relapse ( n = 42) were retrospectively analyzed. Overall, 21.9% of patients were classified as high risk according to the CNS International Prognostic Index (CNS‐IPI). Several biological and clinical features were significantly associated with leptomeningeal involvement, including double‐ or triple‐hit (DHL/THL) status, MYC rearrangement, negative BCL6 expression by IHC, bone marrow involvement, and concomitant R‐SCNSL. Multivariable analysis showed that leptomeningeal involvement independently predicted inferior OS and was associated with a 98% increase in the hazard of death compared with parenchymal relapse (HR = 1.98, 95% CI: 1.20–3.26, p = 0.008). Based on anatomical localization, R‐SCNSL was classified into four subtypes: parenchymal‐only involvement (parenchymal‐CNS [P‐CNS], 68/42%) and parenchymal involvement plus systemic relapse (parenchymal‐concomitant [P‐concomitant], 17/10.5%); and leptomeningeal with or without parenchymal involvement (leptomeningeal‐CNS [LM‐CNS], 52/32.1%) and leptomeningeal with systemic relapse (leptomeningeal‐concomitant [LM‐concomitant], 25/15.4%). This anatomical classification significantly impacted OS and PFS ( p < 0.001). Two‐year OS and PFS were 58.2% and 29.1% for P‐CNS, 32.4% and 17.7% for P‐concomitant, 22% and 13.9% for LM‐CNS, and 7.1% and 0% for LM‐concomitant, respectively. ASCT showed a trend toward improved survival among patients with a response (CR/PR) in a 4‐month landmark analysis. These findings support the clinical application of the anatomical classification in the management of R‐SCNSL.

ABSTRACT Systemic AL amyloidosis is a clonal plasma cell dyscrasia, where toxic free light chains (FLCs) lead to vital organ damage, with risk of early mortality despite effective therapies. Notably, there is a paucity of data on the incidence and predictors of early mortality in the daratumumab (Dara) era. We conducted a multi‐center retrospective cohort study to develop and validate a risk‐prediction model for early mortality (all‐cause death within 6 months) in patients treated with Dara‐based frontline regimens. We used univariate analysis to identify covariates for a logistic regression model predictive of 6‐month mortality and generated a clinical risk score. Among 339 patients across eight centers, 36 (10.6%) experienced early mortality. Multivariate analysis identified four independent predictors: NT‐proBN P > 5300 pg/mL (odds ratio [OR] 3.83, p = 0.006), A ge > 75 years (OR 2.96, p = 0.073), absence of t(11;14) on C ytogenetics (OR 3.0, p = 0.022), and E COG performance status (OR 1.72 per unit increase, p = 0.044). The resulting PACE score stratified patients into three risk groups with a 6‐month mortality of 1.2% (low‐risk), 11.7% (intermediate‐risk), and 50.0% (high‐risk). The PACE score demonstrated superior discrimination (C‐index 0.78) for early mortality compared to Mayo 2004 (0.69) and Mayo 2012 (0.55) staging systems. External validation confirmed robust discrimination (C‐index 0.73) with corresponding 6‐month mortality rates of 4.2%, 15.25%, and 37.04% across respective risk groups. The PACE score provides a clinically applicable tool for early mortality risk stratification in AL amyloidosis patients receiving Dara‐based therapy, and will enable improved patient counseling, clinical trial design, and identification of ultra‐high‐risk patients requiring intensive monitoring and novel therapies.