Synergistic antitumor activity of pan-PI3K inhibition and immune checkpoint blockade in bladder cancer.

BACKGROUND: Immune checkpoint blockade (ICB) induces durable response in approximately 20% of patients with advanced bladder urothelial cancer (aUC). Over 50% of aUCs harbor genomic alterations along the phosphoinositide 3-kinase (PI3K) pathway. The goal of this project was to determine the synergistic effects and mechanisms of action of PI3K inhibition and ICB combination in aUC. METHODS: Alterations affecting the PI3K pathway were examined in The Cancer Genome Atlas (TCGA) and the Cancer Dependency Map databases. Human and mouse cells with Pten deletion were used for in vitro studies. C57BL/6 mice carrying syngeneic tumors were used to determine in vivo activity, mechanisms of action and secondary resistance of pan-PI3K inhibition, ICB and combination. RESULTS: Alterations along the PI3K pathway occurred in 57% of aUCs in TCGA. CRISPR (clustered regularly interspaced short palindromic repeats) knockout of PIK3CA induced pronounced inhibition of cell proliferation (p=0.0046). PI3K inhibition suppressed cancer cell growth, migration and colony formation in vitro. Pan-PI3K inhibition, antiprogrammed death 1 (aPD1) therapy and combination improved the overall survival (OS) of syngeneic mice with PTEN-deleted tumors from 27 days of the control to 48, 37, and 65 days, respectively. In mice with tumors not containing a PI3K pathway alteration, OS was prolonged by the combination but not single treatments. Pan-PI3K inhibition significantly upregulated CD80, CD86, MHC-I, and MHC-II in dendritic cells, and downregulated the transforming growth factor beta pathway with a false discovery rate-adjusted q value of 0.001. Interferon alpha response was significantly upregulated with aPD1 therapy (q value: <0.001) and combination (q value: 0.027). Compared with the control, combination treatment increased CD8+ T-cell infiltration (p=0.005), decreased Treg-cell infiltration (p=0.036), and upregulated the expression of multiple immunostimulatory cytokines and granzyme B (p<0.01). Secondary resistance was associated with upregulation of the mammalian target of rapamycin (mTOR) pathway and multiple Sprr family genes. CONCLUSIONS: The combination Pan-PI3K inhibition and ICB has significant antitumor effects in aUC with or without activated PI3K pathway and warrants further clinical investigation. This combination creates an immunostimulatory tumor milieu. Secondary resistance is associated with upregulation of the mTOR pathway and Sprr family genes

Deep Learning Model for Predicting Immunotherapy Response in Advanced Non-Small Cell Lung Cancer

Importance - Only a small fraction of patients with advanced non−small cell lung cancer (NSCLC) respond to immune checkpoint inhibitor (ICI) treatment. For optimal personalized NSCLC care, it is imperative to identify patients who are most likely to benefit from immunotherapy. Objective - To develop a supervised deep learning−based ICI response prediction method; evaluate its performance alongside other known predictive biomarkers; and assess its association with clinical outcomes in patients with advanced NSCLC. Design, Setting, and Participants - This multicenter cohort study developed and independently validated a deep learning−based response stratification model for predicting ICI treatment outcome in patients with advanced NSCLC from whole slide hematoxylin and eosin–stained images. Images for model development and validation were obtained from 1 participating center in the US and 3 in the European Union (EU) from August 2014 to December 2022. Data analyses were performed from September 2022 to May 2024. Exposure - Monotherapy with ICIs. Main Outcomes and Measures - Model performance measured by clinical end points and objective response rate (ORR) differentiation power vs other predictive biomarkers, ie, programmed death-ligand 1 (PD-L1), tumor mutational burden (TMB), and tumor-infiltrating lymphocytes (TILs). Results - A total of 295 581 image tiles from 958 patients (mean [SD] age, 66.0 [10.6] years; 456 [48%] females and 502 [52%] males) treated with ICI for NSCLC were included in the analysis. The US-based development cohort consisted of 614 patients with median (IQR) follow-up time of 54.5 (38.2-68.1) months, and the EU-based validation cohort, 344 patients with 43.3 (27.4-53.9) months of follow-up. The ORR to ICI was 26% in the developmental cohort and 28% in the validation cohort. The deep learning model’s area under the receiver operating characteristic curve (AUC) for ORR was 0.75 (95% CI, 0.64-0.85) in the internal test set and 0.66 (95% CI, 0.60-0.72) in the validation cohort. In a multivariable analysis, the deep learning model’s score was an independent predictor of ICI response in the validation cohort for both progression-free (hazard ratio, 0.56; 95% CI, 0.42-0.76; P  Conclusions and Relevance - The findings of this cohort study demonstrate a strong and independent deep learning−based feature associated with ICI response in patients with NSCLC across various cohorts. Clinical use of this deep learning model could refine treatment precision and better identify patients who are likely to benefit from ICI for treatment of advanced NSCLC

Germline Variants Associated With Toxicity to Immune Checkpoint Blockade

Immune checkpoint inhibitors (ICIs) have yielded remarkable responses but often lead to immune-related adverse events (irAEs). Although germline causes for irAEs have been hypothesized, no individual variant associated with developing irAEs has been identified. We carried out a genome-wide association study of 1,751 patients on ICIs across 12 cancer types. We investigated two irAE phenotypes: (1) high-grade (3-5) and (2) all-grade events. We identified 3 genome-wide significant associations (P \u3c 5 × 10-8) in the discovery cohort associated with all-grade irAEs: rs16906115 near IL7 (combined P = 3.6 × 10-11; hazard ratio (HR) = 2.1); rs75824728 near IL22RA1 (combined P = 3.5 × 10-8; HR = 1.8); and rs113861051 on 4p15 (combined P = 1.2 × 10-8, HR = 2.0); rs16906115 was replicated in 3 independent studies. The association near IL7 colocalized with the gain of a new cryptic exon for IL7, a critical regulator of lymphocyte homeostasis. Patients carrying the IL7 germline variant exhibited significantly increased lymphocyte stability after ICI initiation, which was itself predictive of downstream irAEs and improved survival

Epigenomic Charting and Functional Annotation of Risk Loci in Renal Cell Carcinoma.Epigenomic Charting and Functional Annotation of Risk Loci in Renal Cell Carcinoma

While the mutational and transcriptional landscapes of renal cell carcinoma (RCC) are well-known, the epigenome is poorly understood. We characterize the epigenome of clear cell (ccRCC), papillary (pRCC), and chromophobe RCC (chRCC) by using ChIP-seq, ATAC-Seq, RNA-seq, and SNP arrays. We integrate 153 individual data sets from 42 patients and nominate 50 histology-specific master transcription factors (MTF) to define RCC histologic subtypes, including EPAS1 and ETS-1 in ccRCC, HNF1B in pRCC, and FOXI1 in chRCC. We confirm histology-specific MTFs via immunohistochemistry including a ccRCC-specific TF, BHLHE41. FOXI1 overexpression with knock-down of EPAS1 in the 786-O ccRCC cell line induces transcriptional upregulation of chRCC-specific genes, TFCP2L1, ATP6V0D2, KIT, and INSRR, implicating FOXI1 as a MTF for chRCC. Integrating RCC GWAS risk SNPs with H3K27ac ChIP-seq and ATAC-seq data reveals that risk-variants are significantly enriched in allelically-imbalanced peaks. This epigenomic atlas in primary human samples provides a resource for future investigation

ATM Deficiency Confers Specific Therapeutic Vulnerabilities in Bladder Cancer

Ataxia-telangiectasia mutated (ATM) plays a central role in the cellular response to DNA damage and ATM alterations are common in several tumor types including bladder cancer. However, the specific impact of ATM alterations on therapy response in bladder cancer is uncertain. Here, we combine preclinical modeling and clinical analyses to comprehensively define the impact of ATM alterations on bladder cancer. We show that ATM loss is sufficient to increase sensitivity to DNA-damaging agents including cisplatin and radiation. Furthermore, ATM loss drives sensitivity to DNA repair-targeted agents including poly(ADP-ribose) polymerase (PARP) and Ataxia telangiectasia and Rad3 related (ATR) inhibitors. ATM loss alters the immune microenvironment and improves anti-PD1 response in preclinical bladder models but is not associated with improved anti-PD1/PD-L1 response in clinical cohorts. Last, we show that ATM expression by immunohistochemistry is strongly correlated with response to chemoradiotherapy. Together, these data define a potential role for ATM as a predictive biomarker in bladder cancer

Genomic landscape of malignnant mesothelioma by site and histology.

8559 Background: Malignnant mesothelioma (MM) is a highly lethal tumor that can develop in the pleura, the peritoneum, the pericardium or the testes. While the genomic features of pleural MM have been well-described overall, less is known about the distribution of genetic alterations (GAs) according to histology. In addition, few reports comparing genetic features according to disease site are available. Methods: We identified patients with pleural or peritoneal mesothelioma with mutational analysis through the GENIE registry. Patient tumor genetic data were provided by Memorial Sloan-Kettering Cancer Center (MSK)-IMPACT and Dana-Farber Cancer Institute (DFCI)-Oncopanel NGS initiatives. Patients with more than one sequenced sample were excluded. We limited our analysis to genes common to all versions of both panels and that were significantly mutated in the TCGA mesothelioma cohort. Mutation and copy number variant (CNV), collectively called GAs, were determined, and were compared using the Fisher’s Exact test and Kruskal-Wallis Test. Comparisons were made both by disease site (pleural vs. peritoneal) and histology for the pleural samples (epithelioid vs. biphasic vs. sarcomatoid). Nominal p-values were obtained, and FDR correction was employed (q&lt;0.1). Results: We identified 439 patients with MM in the GENIE registry who fit the inclusion criteria. The median age was 70.5 years for pleural MM and 60 years for peritoneal MM (Wilcoxon-rank sum test p-value = 3e-9). 72% of patients were male. CDKN2A/CDKN2B GAs (97% and 100% being deletions in CDKN2A and CDKN2B respectively), a described prognostic marker in MM, were more common in pleural than in peritoneal MM. Among pleural MMs, tumors of epithelioid histology had less NF2 GAs than biphasic or sarcomatoid tumors, whereas sarcomatoid tumors had the lowest frequency of BAP1 GAs (Table). Conclusions: Malignnant mesotheliomas of different disease sites and/or histologies display distinct patterns of GAs. These findings may contribute in part to differences in response to treatment and survival among these subsets of MM.[Table: see text] </jats:p

Skeletal Muscle Dysfunction in Experimental Pulmonary Hypertension

Pulmonary arterial hypertension (PAH) is a serious, progressive, and often fatal disease that is in urgent need of improved therapies that treat it. One of the remaining therapeutic challenges is the increasingly recognized skeletal muscle dysfunction that interferes with exercise tolerance. Here we report that in the adult rat Sugen/hypoxia (SU/Hx) model of severe pulmonary hypertension (PH), there is highly significant, almost 50%, decrease in exercise endurance, and this is associated with a 25% increase in the abundance of type II muscle fiber markers, thick sarcomeric aggregates and an increase in the levels of FoxO1 in the soleus (a predominantly type I fiber muscle), with additional alterations in the transcriptomic profiles of the diaphragm (a mixed fiber muscle) and the extensor digitorum longus (a predominantly Type II fiber muscle). In addition, soleus atrophy may contribute to impaired exercise endurance. Studies in L6 rat myoblasts have showed that myotube differentiation is associated with increased FoxO1 levels and type II fiber markers, while the inhibition of FoxO1 leads to increased type I fiber markers. We conclude that the formation of aggregates and a FoxO1-mediated shift in the skeletal muscle fiber-type specification may underlie skeletal muscle dysfunction in an experimental study of PH

<i>TSC1</i>-mutant bladder cancer expression signature in relation to nuclear localization of TFE3 and potential for targetable dependency.

e16532 Background: Mutation and inactivation of the tumor suppressor gene TSC1 is a recurrent (6-10%) event in bladder cancer, but whether it functions as a driver event for tumor development has been uncertain. Methods: We performed differential gene expression and pathway analyses using RNA-seq data from the curated TCGA TSC1 mutant BLCA (n = 26) and TSC1 wild-type BLCA (n = 382) cohort and compared to an internal cohort of putative TSC1/TSC2-driven tumors (n = 63). Mechanistic studies, as well as RNA-seq and H3K27ac ChIP-seq analyses, were conducted in 2 TSC1 mutant/WT BLCA cell lines. Results: Comparison of The Cancer Genome Atlas (TCGA) TSC1-mutant bladder cancers ( TSC1mBLCA) with TCGA TSC1 wildtype tumors ( TSC1WTBLCA) identified a conserved TSC-associated expression signature, similar to ones seen in syndromic TSC tumors. GSEA and DESeq2 analyses implicated both mTORC1 hyperactivation, as well as activation of lysosomal pathways in TSC1mBLCA. We validated our findings by IHC analysis of a separate cohort of TSC1mBLCA (n = 5), compared to TSC1WTBLCA (n = 5). In addition, we found that TFE3, a transcriptional regulator of lysosomal gene expression, was relatively highly expressed in BLCA (compared to other MiT-TFE genes) and was localized to the nucleus in TSC1mBLCA but not in TSC1WTBLCA. Mechanistic studies of two TSC1mBLCA cell lines and their respective TSC1 addback derivatives, recapitulated the phenotype found in human tumors and demonstrated that TFE3 was both post-translationally modified and predominantly nuclear in TSC1-null cell lines compared to TSC1 addbacks. RNA-seq and H3K27ac ChIP-Seq analyses showed that TSC1mBLCA cell lines retained elements of the TSC-associated expression signature that was seen in TSC1mBLCA tumors, confirming differential activation of TFE3 in response to TSC1 loss. Nuclear localization of TFE3 in TSC1mBLCA cell lines was only partially reversed by rapamycin treatment and was unaffected by treatment of Torin1. SiRNA mediated knockdown of TFE3 significantly decreased cell growth and viability in TSC1mBLCA cell lines and did not result in compensatory upregulation of TFEB and MITF. Conclusions: Our findings indicate that TSC1 mutant bladder tumors retain elements of a conserved transcriptional signature that is characterized by nuclear localization and activation of TFE3. Aberrant TFE3 activation likely contributes to TSC1mBLCA development and may therefore be amenable to targeted therapy. </jats:p