Citations

Abstract During the humoral immune response, B cells undergo rapid metabolic reprogramming with a high demand for nutrients, which are vital to sustain the formation of the germinal centers (GCs). Rag-GTPases sense amino acid availability to modulate the mechanistic target of rapamycin complex 1 (mTORC1) pathway and suppress transcription factor EB (TFEB) and transcription factor enhancer 3 (TFE3), members of the microphthalmia (MiT/TFE) family of HLH-leucine zipper transcription factors. However, how Rag-GTPases coordinate amino acid sensing, mTORC1 activation, and TFEB/TFE3 activity in humoral immunity remains undefined. Here, we show that B cell-intrinsic Rag-GTPases are critical for the development and activation of B cells. RagA/RagB deficient B cells fail to form GCs, produce antibodies, and generate plasmablasts in both T-dependent (TD) and T-independent (TI) humoral immune responses. Deletion of RagA/RagB in GC B cells leads to abnormal dark zone (DZ) to light zone (LZ) ratio and reduced affinity maturation. Mechanistically, the Rag-GTPase complex constrains TFEB/TFE3 activity to prevent mitophagy dysregulation and maintain mitochondrial fitness in B cells, which are independent of canonical mTORC1 activation. TFEB/TFE3 deletion restores B cell development, GC formation in Peyer’s patches and TI humoral immunity, but not TD humoral immunity in the absence of Rag-GTPases. Collectively, our data establish Rag-GTPase-TFEB/TFE3 axis as an mTORC1 independent mechanism to coordinating nutrient sensing and mitochondrial metabolism in B cells.

Abstract Mayo Clinic has developed 111 GBM PDXs from newly diagnosed (72 PDX) and recurrent (39 PDX) patients. This large collection of PDXs provides a broad representation of genetic heterogeneity seen in patients. GBM PDXs that perform well in both in vitro and in vivo settings can be extensively utilized in the preclinical setting for downstream analysis. Based on 20 years of experience, we have defined the attributes that best enable preclinical use of our GBM PDX models. Among them are short-term in vitro culturing performance, large scale genomic evaluation data, benchmarked responses to standards of care, and median time to moribund for untreated orthotopic tumors. PDXs with prolonged survival timelines delay study readouts and, in some situations, confound efficacy results due to age-related animal decline. Setting median survival thresholds as part of the upfront experimental design allows for more timely study readouts that are unaffected by these confounding variables. Our preferred cutoff for the Mayo GBM PDX collection is 70 days median survival (n=64). Using this 64 PDX line panel, regimens expected to double survival would produce results within 4-5 months. This timeframe is well within the range of robust health for athymic nude mice. This 64-line collection features both newly diagnosed (nGBM; n=42) and recurrent (rGBM; n=22) tumors. MGMT promoter methylation status was determined by qMS-PCR (25 methylated, 37 unmethylated, 2 indeterminate). Available WES (n=62) and RNAseq (n=42) data, along with our tissue microarray (n=42), are critical resources for biomarker-based evaluations aimed at defining patient populations that are most likely to benefit from a given therapeutic. From these data, the molecular subtypes available include 26 classical, 14 proneural, and 3 mesenchymal. Twenty-seven of these 64 PDX models have also been successfully cultured from fresh as well as cryopreserved cell stocks to further expand their experimental value. Correlative clinical data including diagnosis and treatments are also available (n=61). Current standards of care for GBM include radiation, temozolomide, and bevacizumab. Defining sensitivity to these therapies is an important first step for evaluating the integration of novel therapeutics with these standards. Since the creation of our GBM PDX collection, efficacy of these therapies has been evaluated as follows: RT 26 PDXs, TMZ 20 PDXs, and Avastin 23 PDXs. The Mayo Clinic GBM PDX collection provides an important platform for preclinical evaluation of novel therapeutics. Through careful selection of PDX lines with favorable growth characteristics and known genetic features, researchers can select the most relevant models for their study. This approach will enable more meaningful preclinical work in the hopes of providing more tangible clinical benefits for GBM patients. Citation Format: Danielle M. Burgenske, Ann C. Mladek, Katrina K. Bakken, Zeng Hu, Brett L. Carlson, Paul A. Decker, Jeanette E. Eckel-Passow, Jann N. Sarkaria. Defining critical features for successful preclinical use of glioblastoma patient-derived xenografts (PDX) models for therapeutic assessment and clinical trial development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6897.

Background Nephritis is a common manifestation of IgA vasculitis and is morphologically indistinguishable from IgA nephropathy. While MEST-C scores are predictive of kidney outcomes in IgA nephropathy, their value in IgA vasculitis nephritis has not been investigated in large multiethnic cohorts. Methods Biopsies from 262 children and 99 adults with IgA vasculitis nephritis ( N =361) from 23 centers in North America, Europe, and Asia were independently scored by three pathologists. MEST-C scores were assessed for correlation with eGFR/proteinuria at biopsy. Because most patients ( N =309, 86%) received immunosuppression, risk factors for outcomes were evaluated in this group using latent class mixed models to identify classes of eGFR trajectories over a median follow-up of 2.7 years (interquartile range, 1.2–5.1). Clinical and histologic parameters associated with each class were determined using logistic regression. Results M, E, T, and C scores were correlated with either eGFR or proteinuria at biopsy. Two classes were identified by latent class mixed model, one with initial improvement in eGFR followed by a late decline (class 1, N =91) and another with stable eGFR (class 2, N =218). Class 1 was associated with a higher risk of an established kidney outcome (time to ≥30% decline in eGFR or kidney failure; hazard ratio, 5.84; 95% confidence interval, 2.37 to 14.4). Among MEST-C scores, only E1 was associated with class 1 by multivariable analysis. Other factors associated with class 1 were age 18 years and younger, male sex, lower eGFR at biopsy, and extrarenal noncutaneous disease. Fibrous crescents without active changes were associated with class 2. Conclusions Kidney outcome in patients with biopsied IgA vasculitis nephritis treated with immunosuppression was determined by clinical risk factors and endocapillary hypercellularity (E1) and fibrous crescents, which are features that are not part of the International Study of Diseases of Children classification.

Abstract Radioresistance of melanoma brain metastases limits the clinical utility of conventionally fractionated brain radiation in this disease, and strategies to improve radiation response could have significant clinical impact. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is critical for repair of radiation-induced DNA damage, and inhibitors of this kinase can have potent effects on radiation sensitivity. In this study, the radiosensitizing effects of the DNA-PKcs inhibitor peposertib were evaluated in patient-derived xenografts of melanoma brain metastases (M12, M15, M27). In clonogenic survival assays, peposertib augmented radiation-induced killing of M12 cells at concentrations ≥100 nmol/L, and a minimum of 16 hours exposure allowed maximal sensitization. This information was integrated with pharmacokinetic modeling to define an optimal dosing regimen for peposertib of 125 mpk dosed just prior to and 7 hours after irradiation. Using this drug dosing regimen in combination with 2.5 Gy × 5 fractions of radiation, significant prolongation in median survival was observed in M12-eGFP (104%; P = 0.0015) and M15 (50%; P = 0.03), while more limited effects were seen in M27 (16%, P = 0.04). These data support the concept of developing peposertib as a radiosensitizer for brain metastases and provide a paradigm for integrating in vitro and pharmacokinetic data to define an optimal radiosensitizing regimen for potent DNA repair inhibitors.

Context.— The Nottingham Grading System (NGS) developed by Elston and Ellis is used to grade invasive breast cancer (IBC). Glandular (acinar)/tubule formation is a component of NGS. Objective.— To investigate the ability of pathologists to identify individual structures that should be classified as glandular (acinar)/tubule formation. Design.— A total of 58 hematoxylin-eosin photographic images of IBC with 1 structure circled were classified as tubules (41 cases) or nontubules (17 cases) by Professor Ellis. Images were sent as a PowerPoint (Microsoft) file to breast pathologists, who were provided with the World Health Organization definition of a tubule and asked to determine if a circled structure represented a tubule. Results.— Among 35 pathologists, the κ statistic for assessing agreement in evaluating the 58 images was 0.324 (95% CI, 0.314–0.335). The median concordance rate between a participating pathologist and Professor Ellis was 94.1% for evaluating 17 nontubule cases and 53.7% for 41 tubule cases. A total of 41% of the tubule cases were classified correctly by less than 50% of pathologists. Structures classified as tubules by Professor Ellis but often not recognized as tubules by pathologists included glands with complex architecture, mucinous carcinoma, and the “inverted tubule” pattern of micropapillary carcinoma. A total of 80% of participants reported that they did not have clarity on what represented a tubule. Conclusions.— We identified structures that should be included as tubules but that were not readily identified by pathologists. Greater concordance for identification of tubules might be obtained by providing more detailed images and descriptions of the types of structures included as tubules.

Background: Anaplastic large cell lymphomas (ALCLs) are CD30-positive T-cell lymphomas that share pathologic features but differ in clinical presentation, outcome, and molecular features. The World Health Organization (WHO) and International Consensus Classification (ICC) classify ALCLs by presence or absence of ALK rearrangements (R) and clinical presentation (systemic, cutaneous [c], or breast implant-associated [BIA]). ICC, but not WHO, recognizes DUSP22-R as defining a new genetic subtype of ALK- ALCL. The classifications otherwise do not reflect additional molecular heterogeneity in genetics (e.g., TP63-R) or therapeutic vulnerabilities (e.g., JAK-STAT3 pathway activation). Methods: ALCLs (N=689) underwent expert consensus review (WHO/ICC) through the Lymphoma/Leukemia Molecular Profiling Project (LLMPP). All cases also underwent genetic subtyping (ALK, DUSP22, TP63, and triple-negative [TN]) using fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), as well as IHC for phospho-STAT3 Tyr705 (pSTAT3). RNAseq was performed and evaluable in 393 cases; the remaining cases had insufficient tissue, tumor content, RNA yield or quality, and/or sequencing data quality. Sequenced and non-sequenced sub-cohorts had similar demographics and subtype distribution. Results: Unsupervised gene expression profiling (GEP) identified 2 main molecular types of ALCL. Type I ALCLs predominantly included ALK+ ALCLs, BIA-ALCLs, and a subset of TN ALCLs (designated TN-I), whereas Type II ALCLs predominantly included ALCLs with DUSP22-R, TP63-R, or both (double-hit; DH), and the remaining TN ALCLs (TN-II). Type I ALCLs were strongly associated with pSTAT3 expression (74.2±26.4% positive malignant cells vs 9.9±22.7% for Type II; P<0.0001). An independently derived pSTAT3 staining threshold of 30% assigned Type I vs II with 91% accuracy. A third cluster of ALCLs, mostly cALCL, showed an epithelial GEP signature rather than a lymphoma signature; these cases were assigned to Types I or II based on pSTAT3 IHC. Distinct sub-signatures were identified for ALK+, DUSP22-R, TP63-R, and BIA ALCL (Fig. 1), but not ALK- ALCL or cALCL. Gene set enrichment analysis showed Type I ALCLs to be enriched for JAK-STAT3 signaling genes (normalized enrichment score [NES], 2.22; FDR<0.0001) and related pathways, such as TNFα-NFκB signaling (NES, 2.21; FDR<0.0001). In contrast, Type II ALCLs were enriched for cell cycle, DNA repair, epigenetic, and metabolic pathway genes, but not for major tyrosine kinase-mediated signaling pathway genes. Enriched epigenetic pathways included chromatin modifying enzymes (NES, -1.86; FDR=0.002) and histone methylation (NES, -1.71; FDR=0.002). EZH2 was the most overexpressed gene in Type II ALCLs (fold-change, 7.74; FDR=8.84×10 -306). At the protein level, EZH2 IHC H-scores were 275±45 in Type II and 168±70 in Type I ALCLs (P<0.0001); H3K27me3 H-scores were 170±81 and 76±63, respectively (P<0.001). The top metabolic gene set involved cholesterol biosynthesis (NES, -1.88; FDR=0.002). Overall survival (OS) data were available in 257 systemic ALCL patients (145 sequenced and 112 non-sequenced; cALCL and BIA-ALCL were excluded). Non-sequenced TN ALCLs were assigned to TN-I or TN-II using pSTAT3 IHC. The results supported earlier data indicating favorable prognosis of DUSP22-R ALCL (5 y OS, 95%) and ALK+ ALCL (87%), intermediate prognosis of TN ALCL (TN-I, 52% and TN-II, 38%; P=NS), and poor prognosis of TP63-R/DH ALCL (0%)(P<0.0001; Fig. 2). Conclusions: Two overarching molecular types of ALCL exist, predominantly associated with the presence (Type I) or absence (Type II) of the JAK-STAT3 signaling program. Distinct GEP signatures exist for ALK+ ALCL and BIA-ALCL (predominantly Type I), and DUSP22-R ALCL and TP63-R ALCL (predominantly Type II). TN ALCLs lacking ALK-R, DUSP22-R, and TP63-R can be stratified into TN-I and TN-II subtypes. pSTAT3 IHC has >90% accuracy as a surrogate for GEP-based subtyping. ALK- ALCL and cALCL cluster by molecular subtype rather than by defining GEP signatures. Type II ALCLs are enriched for targetable epigenetic and metabolic pathways, including EZH2/histone methylation and cholesterol biosynthesis. This molecular classification is diagnostically, prognostically, and potentially therapeutically relevant, and can be applied using FISH and IHC in routine practice and in the clinical trial setting.

Abstract Radiation therapy (RT) is an important non-surgical clinical treatment for glioblastoma (GBM) and brain metastases, but inherent radio-resistance can limit the efficacy in many patients. The Ataxia Telangiectasia Mutated (ATM) protein critically regulates radiotherapy-mediated DNA damage repair pathways, and defects in this kinase can lead to profound radiosensitivity. In this study, the efficacy of the highly potent ATM inhibitor WSD0628 was evaluated in GBM and melanoma brain metastasis patient derived xenografts (PDXs). In short term explant cultures of three PDXs, WSD0628 robustly suppressed RT-induced autophosphorylation of ATM at serine 1981 and ATM-mediated phosphorylation of Chk2 at threonine-68 and KAP1 at serine-824, with maximal inhibition at 100 nM. Similarly, RT-induced gH2AX foci were significantly reduced when combined with WSD0628 in the GBM43 cells. Consistent with the importance of ATM in the DNA damage response after radiation, WSD0628 significantly increased the radiosensitivity of U251 cells in clonogenic survival assays (0nM vs. 30nM at 5Gy, p<0.01). To evaluate the optimal duration of drug exposure, WSD0628 was removed from U251 cells at various intervals following irradiation with 5 Gy. Using clonogenic survival as a readout, a 10-fold increase in cytotoxicity was observed with an 8 hour drug exposure, with progressively increasing cytotoxicity gains with exposures up to 24 hours. In vivo efficacy of WSD0628 was evaluated in multiple intracranial PDX models. In an initial dose-ranging study, robust radiosensitization was observed in GBM43 treated with 2 Gy x 5 fractions combined with 5 mg/kg WSD0628 (20 day survival extension) or 10mg/kg WSD0628 (39 day with 10 mg/kg; p<0.01 for both dose levels). Moreover, a single dose of 12.5 Gy combined with 10 mg/kg WSD0628 had profound impact on treatment efficacy in the melanoma brain metastasis M12 PDX: at 180 days post-treatment, all mice in the combination group were electively euthanized, while median survival for sham or RT alone was 17d and 49d, respectively. Histologic analysis identified large intracranial tumors in all sham and RT-only treated mice, but only a small accumulation of melanotic cells without obvious tumor in the combination-treated mice. Immunohistochemical staining of NeuN and GFAP in the combination-treated mice showed preserved neuronal density at the 180-post treatment timepoint and minimal reactive gliosis within the ‘tumor scar’. An ongoing study is comparing this single-fraction 12.5 Gy regimen to a 2.4 Gy x 10 fraction regimen alone or in combination with WSD0628. Collectively, this study demonstrates the potential for profound radiosensitizing effects of WSD0628 in combination without obvious neuronal toxicity and has provided the scientific rational for the first-in-man study of this combination in recurrent GBM (NCT05917145) at Mayo Clinic. Citation Format: Zhiyi Xue, Ann C. Mladek, Sneha Rathi, Danielle M. Burgenske, Shiv K. Gupta, Brett L. Carlson, Zeng Hu, Lauren L Ott, Katrina K. Bakken, Rachael A. Vaubel, William F Elmquist, Wei Zhong, Jann N Sarkaria. The radiosensitizing effects of the novel brain penetrant and potent ATM inhibitor WSD0628 in glioblastoma and melanoma patient derived xenografts [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A148.

Abstract Large PDX collections provide broad representation of tumor genetics and are commonly used to evaluate the clinical potential of novel therapeutic strategies. In many solid malignancies, profound benefit in PDX preclinical studies has foreshadowed eventual FDA approval of clinically impactful therapeutics. With a lack of corresponding progress in glioblastoma (GBM), the value of PDX testing has been questioned. Mayo Clinic has developed 111 GBM PDXs from newly diagnosed (72 PDX) and recurrent (39 PDX) patients, and over the past 23 years, we have used this collection for extensive in vivo preclinical testing of 145 drugs in both orthotopic and heterotopic settings. As part of this work, the impact of standard of care therapy with temozolomide (TMZ) and bevacizumab was evaluated in orthotopic models using clinically relevant dosing regimens. For each animal study, the ratio of median survival for treated vs. control animals were used to define a ‘survival ratio’ as a normalized metric of benefit. TMZ efficacy was evaluated in 26 newly diagnosed GBM PDXs, with equal representation of MGMT methylated and unmethylated tumors. As expected, MGMT methylated PDXs were much more sensitive to TMZ as compared to unmethylated PDXs (median survival ratio 4.47 vs 1.48, respectively), and similar to clinical experience, 2 MGMT unmethylated PDXs were markedly sensitive to TMZ, while 1 MGMT methylated PDX was relatively resistant. In a more limited study, dose-dense vs. standard TMZ dosing was compared in 7 PDXs (4 MGMT methylated, 3 MGMT unmethylated). In a pooled analysis of all lines, both standard (HR=0.09; 95% CI: 0.06–0.14) and dose-dense TMZ therapy (HR=0.09; 95% CI: 0.06–0.14) were superior to vehicle controls (p<0.001). Similar to the clinical results in the Phase III randomized RTOG 0525 trial, survival with the dose-dense TMZ was no different than standard dosing (p=0.88, HR=0.98; 95% CI: 0.72-1.32). In an analysis of bevacizumab across 32 PDXs, mice were dosed once a week until moribund, and a minimum overall median survival benefit of 20% was used to define ‘responders’. Thirteen PDXs met this criterion, although the overall survival ratio among this subset was limited (range 1.21-1.65). Only 2 models had a 50% or greater survival benefit. Stratification by disease state (24 newly diagnosed vs 8 recurrent) showed equivalent performance (median survival benefit 1.13 for both). These results are consistent with the limited survival benefit observed with bevacizumab and the RTOG 0825 clinical trial. In summary, the results from these relatively inexpensive PDX studies demonstrate the potential of pre-clinical PDX trials to evaluate the extent of benefit and fraction of responsive PDXs to a novel therapeutic. If analyzed across an ‘adequate’ number of PDXs, these results potentially can be used to identify predictive biomarkers or to compare various treatment regimens prior to clinical testing. Citation Format: Danielle M Burgenske, Ann C Mladek, Katrina K Bakken, Zeng Hu, Brett L Carlson, Paul A Decker, Jeanette E Eckel-Passow, Jann N Sarkaria. The utility of pre-clinical trials in glioblastoma patient-derived xenografts (PDXs) models for informing clinical trial development of therapeutic strategies [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A002.

The scarcity of liver grafts has prompted developments in living donor liver transplantations (LDLT), with previous literature illustrating similar outcomes in recipients compared to deceased donor transplants. However, significant concerns regarding living donor morbidity and mortality have yet to be examined comprehensively. This study aims to provide estimates of the incidence of various outcomes in living liver donors. In this meta-analysis, Medline and Embase were searched from inception to July 2022 for articles assessing the incidence of outcomes in LDLT donors. Complications in the included studies were classified into respective organ systems. Analysis of incidence was conducted using a generalized linear mixed model with Clopper-Pearson intervals. Eighty-seven articles involving 60,829 living liver donors were included. The overall pooled incidence of complications in LDLT donors was 24.7% (CI: 21.6%–28.1%). The incidence of minor complications was 17.3% (CI: 14.7%–20.3%), while the incidence of major complications was lower at 5.5% (CI: 4.5%–6.7%). The overall incidence of donor mortality was 0.06% (CI: 0.0%–0.1%) in 49,027 individuals. Psychological complications (7.6%, CI: 4.9%–11.5%) were the most common among LDLT donors, followed by wound-related (5.2%, CI: 4.4%–6.2%) and respiratory complications (4.9%, CI: 3.8%–6.3%). Conversely, cardiovascular complications had the lowest incidence among the subgroups at 0.8% (CI: 0.4%–1.3%). This study presents the incidence of post-LDLT outcomes in living liver donors, illustrating significant psychological, wound-related, and respiratory complications. While significant advancements in recent decades have contributed towards decreased morbidity in living donors, our findings call for targeted measures and continued efforts to ensure the safety and quality of life of liver donors post-LDLT.