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PURPOSE Advances in biological measurement technologies are enabling large-scale studies of patient cohorts across multiple omics platforms. Holistic analysis of these data can generate actionable insights for translational research and necessitate new approaches for data integration and mining. METHODS We present a novel approach for integrating data across platforms on the basis of the shared nearest neighbors algorithm and use it to create a network of multiplatform data from the immunogenomic profiling of non–small-cell lung cancer project. RESULTS Benchmarking demonstrates that the shared nearest neighbors-based network approach outperforms a traditional gene-gene network in capturing established interactions while providing new ones on the basis of the interplay between measurements from different platforms. When used to examine patient characteristics of interest, our approach provided signatures associated with and new leads related to recurrence and TP53 oncogenotype. CONCLUSION The network developed offers an unprecedented, holistic view into immunogenomic profiling of non–small-cell lung cancer, which can be explored through the accompanying interactive browser that we built.

Synergy between B02 and AZD1775 is associated with impaired Rad51-mediated homologous recombination repair in HPV-positive HNSCC cells

Supplementary Table S1

Supplementary Materials and Methods

Despite advances in surgery, chemotherapy, and radiation, there are limited treatment options for advanced head and neck squamous cell carcinoma (HNSCC) and survival remains very poor. Therefore, effective therapies are desperately needed. Recently, selective exploitation of DNA damage and replication stress responses has become a novel approach for cancer treatment. Wee1 kinase and Rad51 recombinase are two proteins involved in regulating replication stress and homologous recombination repair in cancer cells. In this study, we investigated the combined effect of Rad51 inhibitor (B02) and Wee1 inhibitor (AZD1775) in vitro and in vivo in various HNSCC cell lines. Clonogenic survival assays demonstrated that B02 synergized with AZD1775 in vitro in all HNSCC cell lines tested. The synergy between these drugs was associated with forced CDK1 activation and reduced Chk1 phosphorylation leading to induction of excessive DNA damage and replication stress, culminating in aberrant mitosis and apoptosis. Our results showed that elevated Rad51 mRNA expression correlated with worse survival in HNSCC patients with HPV-positive tumors. The combination of B02 and AZD1775 significantly inhibited tumor growth in vivo in mice bearing HPV-positive HNSCC tumors as compared to HPV-negative HNSCC. This differential sensitivity appears to be linked to HPV-positive tumors having more in vivo endogenous replication stress owing to transformation by E6 and E7 oncogenes. Furthermore, addition of B02 radiosensitized the HPV-negative HNSCC tumors in vitro and in vivo. In conclusion, our data implicate that a novel rational combination with Rad51 and Wee1 inhibitors holds promise as synthetic lethal therapy, particularly in high-risk HPV-positive HNSCC.

Single agent activity of B02 in HNSCC cell lines, Cell growth assay in HNSCC cells, Cell growth assay in HNSCC cells, and Synergy between B02 and AZD1775 is mediated in part through forced activation of CDK1 and inhibition of Chk1 associated with impaired Rad51-mediated homologous recombination repair in HNSCC cells.

Table S2. Immunological gene signatures enriched in multiple cancer types.

Supplementary figures and figure legends Supplementary Figure 1. mTOR inhibitors do not impair homologous recombination repair in non-transformed breast epithelial cells. Supplementary Figure 2. mTOR inhibitors do not affect expression of key HR-related proteins in BRCA-proficient TNBCs. Supplementary Figure 3. Microarray and Ingenuity Pathway Analysis (IPA).

Purpose:Salivary gland adenoid cystic carcinoma (ACC) has heterogeneous clinical behavior. Currently, all patients are treated uniformly, and no standard-of-care systemic therapy exists for metastatic ACC. We conducted an integrated proteogenomic analyses of ACC tumors to identify dysregulated pathways and propose a classification with therapeutic implications.Experimental Design:RNA/DNA sequencing of 54 flash-frozen salivary ACCs and reverse phase protein array (RPPA) in 38 specimens were performed, with validation by Western blotting and/or IHC. Three independent ACC cohorts were used for validation.Results:Both unbiased RNA sequencing (RNA-seq) and RPPA analysis revealed two molecular subtypes: ACC-I (37%) and ACC-II (63%). ACC-I had strong upregulation of MYC, MYC target genes, and mRNA splicing, enrichment of NOTCH-activating mutations, and dramatically worse prognosis. ACC-II exhibited upregulation of TP63 and receptor tyrosine kinases (AXL, MET, and EGFR) and less aggressive clinical course. TP63 and MYC were sufficient to assign tumors to ACC subtypes, which was validated in one independent cohort by IHC and two additional independent cohorts by RNA-seq. Furthermore, IHC staining for MYC and P63 protein levels can be used to identify ACC subtypes, enabling rapid clinical deployment to guide therapeutic decisions. Our data suggest a model in which ACC-I is driven by MYC signaling through either NOTCH mutations or direct amplification, which in turn suppress P63 signaling observed in ACC-II, producing unique therapeutic vulnerabilities for each subtype.Conclusions:Cooccurrence of multiple actionable protein/pathways alterations in each subtype indicates unique therapeutic vulnerabilities and opportunities for optimal combination therapy for this understudied and heterogeneous disease.

Supplementary Figures 1-10. Figure-S1. Immune score prediction of response to PD-1 immune checkpoint blockade; Figure-S2. Immune mutation burden in cancer; Figure-S3. Transcriptome alteration of immunome in cancer; Figure-S4. Enrichment of differentially expressed genes (DEGs) in immunological gene signatures; Figure-S5. Immune infiltration score in cancer; Figure-S6. Candidate immune responder genes in cancer; Figure-S7. The ROC curves for different signatures in two datasets; Figure-S8. Barplots showing the AUCs of different signatures across 17 datasets; Figure-S9. CNV alteration and QTL of immune-related genes; Figure-S10. QTLs of immune-related genes across cancer types.

Alterations in immune-related pathways are common hallmarks of cancer. A comprehensive understanding of how cancer mutations rewire immune signaling networks and functional output across cancer types is instrumental to realize the full potential of immunotherapy. Here, we systematically interrogated somatic mutations involved in immune signaling that alter immune responses in patients with cancer. To do so, we developed a Network-based Integrative model to Prioritize Potential immune respondER genes (NIPPER). Identified mutations were enriched in essential protein domains and genes identified by NIPPER were associated with responsiveness to multiple immunotherapy modalities. These genes were used to devise an interactome network propagation framework integrated with drug-associated gene signatures to identify potential immunomodulatory drug candidates. Together, our systems-level analysis results help interpret the heterogeneous immune responses among patients and serve as a resource for future functional studies and targeted therapeutics.Significance:This study demonstrates that integration of multi-omics data can help identify critical molecular determinants for effective targeted therapeutics.

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Supplementary Figures S1-S5. NPRL2/TUSC4 up-regulation doesn't correlate with patient survival (S1); Microarray analysis of NPRL2/TUSC4 knockdown cells (S2); The expression of NPRL2/TUSC4-deficient genes are correlated with canonical pathways changes (S3); Histogram of cell cycle distribution for control and NPRL2/TUSC4-deficiency cells (S4); Colony formation assay of NPRL2/TUSC4 overexpression and NPRL2/TUSC4 knockdown cells (S5).

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In precancerous and cancerous lesions, excessive growth signals resulting from activation of oncogenes or loss of tumor suppressor genes lead to intensive replication stress, which is recognized by a high level of replication-associated DNA double-strand breaks (DSB). However, the molecular mechanism by which cells alleviate excessive replication stress remains unclear. In this study, we report that the human nuclease/helicase DNA2 facilitates homologous recombination to repair replication-associated DNA DSBs, thereby providing cells with survival advantages under conditions of replication stress. The nuclease activity of DNA2 was required for DSB end resection, which allowed subsequent recruitment of RPA and RAD51 to repair DSBs and restart replication. More importantly, DNA2 expression was significantly increased in human cancers and its expression correlated with patient outcome. Our findings therefore indicate that enhanced activity of DSB resection likely constitutes one mechanism whereby precancerous and cancerous cells might alleviate replication stress. Cancer Res; 72(11); 2802–13. ©2012 AACR.

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Genome-wide sequencing studies in breast cancer have recently identified frequent mutations in the zinc finger protein 668 (ZNF668), the function of which is undefined. Here, we report that ZNF668 is a nucleolar protein that physically interacts with and regulates p53 and its negative regulator MDM2. Through MDM2 binding, ZNF668 regulated autoubiquitination of MDM2 and its ability to mediate p53 ubiquitination and degradation. ZNF668 deficiency also impaired DNA damage–induced stabilization of p53. RNA interference–mediated knockdown of ZNF668 was sufficient to transform normal mammary epithelial cells. ZNF668 effectively suppressed breast cancer cell proliferation in vitro and tumorigenicity in vivo. Taken together, our studies identify ZNF668 as a novel breast tumor suppressor gene that functions in regulating p53 stability. Cancer Res; 71(20); 6524–34. ©2011 AACR.

PDF file - 633K, Supplementary Fig 1: DNA2 associates with replication forks and prevents the accumulation of replication-associated DSBs. S. Fig 2: DNA2 facilitates HR repair. S. Fig 3: DNA2's function in HR repair is dependent on its nuclease activity. S. Fig 4: Biological effects of DNA2 depletion on cancer cells. S. Fig 5: Effects of DNA2 depletion on the generation of ROS. PLUS supplemental Materials and Methods

Legend for Supplementary Figures S1-S5.

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PDF file - 444K, S1 TOP2A transcript levels in ovarian and lung cancers stratified by DLX4 expression. S2 Overexpression and knockdown of TOP2. S3 DLX4 does not stimulate HR-mediated repair of DSBs. S4 DLX4 increases the magnitude of erroneous end-joining repair of DSBs.

SUMMARYSince the discovery of BRCA1 and BRCA2 mutations as cancer risk factors, we have gained substantial insight into their role in maintaining genomic stability through homologous recombination (HR) DNA repair. However, upon pan-cancer analysis of tumors from The Cancer Genome Atlas (TCGA), we found that mutations in BRCA1/2 and other classical HR genes only identified 10-20% of tumors that display genomic evidence of HR deficiency (HRD), suggesting that the cause of the vast majority of HR defects in tumors is unknown. As HRD both predisposes individuals to cancer development and leads to therapeutic vulnerabilities, it is critical to define the spectrum of genetic events that drive HRD. Here, we employed a network-based approach leveraging the abundance of molecular characterization data from TCGA to identify novel drivers of HRD. We discovered that over half of putative genes driving HRD originated outside of canonical DNA damage response genes, with a particular enrichment for RNA binding protein (RBP)-encoding genes. These novel drivers of HRD were cross-validated using an independent ICGC cohort, and were enriched in GWAS loci associated with cancer risk. Experimental approaches validated over 90% of our predictions in a panel of 50 genes tested by siRNA and 31 additional engineered mutations identified from TCGA patient tumors. Moreover, genetic suppression of identified RBPs or pharmacological inhibition of RBPs induced PARP inhibition. Further mechanistic studies indicate that some RBPs are recruited to sites of DNA damage to facilitate repair, whereas others control the expression of canonical HR genes. Overall, this study greatly expands the repertoire of known drivers of HRD and their contributions to DNA damage repair, which has implications for not only future mechanistic studies, but also for genetic screening and therapy stratification.HIGHLIGHTSThe majority of HR deficiencies detected cannot be directly attributed to aberrations in canonical HR genes.Integrated network analysis identifies RNA binding proteins (RBPs) as a novel driver of HR deficiency in patient tumors.RBP dysfunction can produce HR deficiencies through both dysregulation of canonical HR genes and action at sites of DNA damage.

TP53 mutations are the most common cancer driver mutations among all cancers. Although some TP53 mutations lead to a loss of function of wild-type p53, many other TP53 mutations confer gain-of-function (GOF) activities, which promote cancer cell metastasis and pro-tumorigenic inflammation. Despite that many functional models of GOF mutant p53 (mutp53) have been proposed previously, the mechanisms involved in mutp53 GOF still remain largely elusive. Here we show that by directly targeting minichromosome maintenance complex component 5 (MCM5), a component of the hexametric DNA helicase MCM2-7 complex, GOF mutp53 predisposes cancer cells to replication stress and chromosomal instability, which leads to a tumor cell-autonomous and stimulator of interferon genes (STING)-dependent cytosolic DNA response that activates downstream non-canonical nuclear factor kappa light chain enhancer of activated B cell (NC-NF-κB) signaling. Furthermore, our results demonstrate that GOF mutp53-activated tumor cell-intrinsic STING-NC-NF-κB signaling not only stimulates tumor cell metastasis, but also promotes tumor immune resistance through fostering an immunosuppressive tumor microenvironment. Therefore, our findings that mutp53 exerts its GOF role through pro-tumorigenic MCM5-CIN-STING-NC-NF-κB signaling highlight the importance of TP53 and its inactivation in cancer genome evolution of genomic instability that drives tumor development and progression. Citation Format: Mei Zhao, Tianxiao Wang, Frederico O. Gleber-Netto, Zhen Chen, Daniel J. McGrail, Javier A. Gomez, Wutong Ju, Mayur A. Gadhikar, Wencai Ma, Li Shen, Ximing Tang, Sen Pathak, Maria G. Raso, Jared Burks, Shiaw-Yih Lin, Jing Wang, Asha S. Multani, Curtis R. Pickering, Junjie Chen, Jeffrey N. Myers, Ge Zhou. Mutant p53 gains oncogenic functions through a cytosolic DNA response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2585.

Purpose: Salivary gland adenoid cystic carcinoma (ACC) has heterogeneous clinical behavior. Currently, all patients are treated uniformly, and no standard-of-care systemic therapy exists for metastatic ACC. We conducted an integrated proteogenomic analyses of ACC tumors to identify dysregulated pathways and propose a classification with therapeutic implications. Experimental Design: RNA/DNA sequencing of 54 flash-frozen salivary ACCs and reverse phase protein array (RPPA) in 38 specimens were performed, with validation by Western blotting and/or IHC. Three independent ACC cohorts were used for validation. Results: Both unbiased RNA sequencing (RNA-seq) and RPPA analysis revealed two molecular subtypes: ACC-I (37%) and ACC-II (63%). ACC-I had strong upregulation of MYC, MYC target genes, and mRNA splicing, enrichment of NOTCH-activating mutations, and dramatically worse prognosis. ACC-II exhibited upregulation of TP63 and receptor tyrosine kinases (AXL, MET, and EGFR) and less aggressive clinical course. TP63 and MYC were sufficient to assign tumors to ACC subtypes, which was validated in one independent cohort by IHC and two additional independent cohorts by RNA-seq. Furthermore, IHC staining for MYC and P63 protein levels can be used to identify ACC subtypes, enabling rapid clinical deployment to guide therapeutic decisions. Our data suggest a model in which ACC-I is driven by MYC signaling through either NOTCH mutations or direct amplification, which in turn suppress P63 signaling observed in ACC-II, producing unique therapeutic vulnerabilities for each subtype. Conclusions: Cooccurrence of multiple actionable protein/pathways alterations in each subtype indicates unique therapeutic vulnerabilities and opportunities for optimal combination therapy for this understudied and heterogeneous disease.

BackgroundAdoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TIL) yielded clinical benefit in patients with checkpoint blockade immunotherapy-refractory non-small cell lung cancer (NSCLC) prompting a renewed interest in TIL-ACT. This preclinical study explores the feasibility of producing a NSCLC TIL product with sufficient numbers and enhanced attributes using an improved culture method.MethodsTIL from resected NSCLC tumors were initially cultured using (1) the traditional method using interleukin (IL)-2 alone in 24-well plates (TIL 1.0) or (2) IL-2 in combination with agonistic antibodies against CD3 and 4-1BB (Urelumab) in a G-Rex flask (TIL 3.0). TIL subsequently underwent a rapid expansion protocol (REP) with anti-CD3. Before and after the REP, expanded TIL were phenotyped and the complementarity-determining region 3 β variable region of the T-cell receptor (TCR) was sequenced to assess the T-cell repertoire.ResultsTIL 3.0 robustly expanded NSCLC TIL while enriching for CD8+ TIL in a shorter manufacturing time when compared with the traditional TIL 1.0 method, achieving a higher success rate and producing 5.3-fold more TIL per successful expansion. The higher proliferative capacity and CD8 content of TIL 3.0 was also observed after the REP. Both steps of expansion did not terminally differentiate/exhaust the TIL but a lesser differentiated population was observed after the first step. TIL initially expanded with the 3.0 method exhibited higher breadth of clonotypes than TIL 1.0 corresponding to a higher repertoire homology with the original tumor, including a higher proportion of the top 10 most prevalent clones from the tumor. TIL 3.0 also retained a higher proportion of putative tumor-specific TCR when compared with TIL 1.0. Numerical expansion of TIL in a REP was found to perturb the clonal hierarchy and lessen the proportion of putative tumor-specific TIL from the TIL 3.0 process.ConclusionsWe report the feasibility of robustly expanding a T-cell repertoire recapitulating the clonal hierarchy of the T cells in the NSCLC tumor, including a large number of putative tumor-specific TIL clones, using the TIL 3.0 methodology. If scaled up and employed as a sole expansion platform, the robustness and speed of TIL 3.0 may facilitate the testing of TIL-ACT approaches in NSCLC.

A defective homologous recombination (HR) repair program increases tumor incidence as well as providing a survival advantage in patients with breast and ovarian cancers. Here, we hypothesize that the tumor-promoting side of genome maintenance programs may be contributed by a self-renewal protein, nucleostemin (NS). To address this issue, we established its functional importance in mammary tumor progression in mice and showed that mammary tumor cells become highly susceptible to replicative DNA damage following NS depletion and are protected from hydroxyurea-induced damage by NS overexpression. Breast cancer cells with basal-like characters display more reliance on NS for genome maintenance than those with luminal characters. Mechanistically, NS-deficient cells demonstrate a significantly reduced HR repair activity. TCGA analyses of human breast cancers revealed that NS is co-enriched positively with HR repair proteins and that high NS expression correlates with low HR defects and predicts poor progression-free survival and resistance to knockdown of cell cycle checkpoint genes in triple-negative/basal-like breast cancers. This work indicates that NS constitutes a tumor-promoting genome maintenance program required for mammary tumor progression.

6090 Background: ACC is a common salivary gland malignancy for which there is no FDA approved therapies. Despite a quiet genome, ACC has significant tumoral heterogeneity, which may facilitate the metastatic relapse. Studies focusing on the deep profile of ACC tumor microenvironment (TME) are lacking. Here we explored the TME of ACC using imaging mass cytometry (IMC) and assessed TME attributes and its association with histology and clinical outcomes. Methods: Two tissue microarrays from 62 ACC patients (pts) were built and stained with 37 metal-tagged markers. IMC was performed with the Fluidigm Helios CyTOF instrument utilizing the Hyperion Imaging System Laser ablation. Tissue and cell segmentation and multiplex imaging analysis were performed with Visiopharm software using pre-trained artificial intelligence algorithms. Comparison of cell types and markers densities among histology sub-groups were analyzed with Wilcoxon signed-rank test and its association with overall survival (OS) with log-rank and Cox Proportional Hazards. Results: Of 62 pts, 37% (23/62) had solid histology, 19% (12/62) were metastatic at diagnosis, and 55% (30/54) had disease recurrence during follow-up. With a median follow-up of 7.9 y, the median OS was 9.3y (CI 95%, 7.8-NR). Pts with solid subtype had poorer OS than non-solid histology (5.2 vs 14.6 y, p = 0.004). The IMC final dataset comprised 507,524 single-cells. No significant differences were found in cell subpopulations density between solid and non-solid histology. The most represented cell population in the stroma were macrophages, followed by CD8+ T cells, and fibroblasts. A higher percentage of M2-macrophage was associated with poor survival (p = 0.001), whereas, a higher percentage of M1-macrophage (M1:M2 ratio) was associated with better prognosis. No other immune cell type, fibroblasts or immune cell functional markers (TIGIT, TIM3, granzyme B, and PD-L1) correlated with survival. Regarding tumor markers, a higher expression of BCL-2, B7-H4, and Ki67 in the tumor cells were associated with worst survival; and remained statistically associated after adjustment for histology and staging (all p < 0.001). Solid histology had a significantly higher density of tumor cells expressing B7-H4, BCL-2, and Ki67 compared to cribriform and tubular histology. A higher expression of myoepithelial marker (p63+) were associated with a better survival when compared with tumors with low p63 expression. Conclusions: ACC’s TME is composed mainly of macrophages. Despite having no significant differences in cellular composition, a higher density of tumor cells expressing BCL-2, B7-H4 and Ki67 were found in the solid histology and these markers were independent predictors of poor prognosis. The overexpression of BCL-2 and B7-H4 in the solid histology provides a scientific rationale for BCL-2 and/or B7-H4 targeting for the most aggressive ACC.

Nucleotide excision repair (NER) resolves DNA adducts, such as those caused by ultraviolet light. Deficient NER (dNER) results in a higher mutation rate that can predispose to cancer development and premature ageing phenotypes. Here, we used isogenic dNER model cell lines to establish a gene expression signature that can accurately predict functional NER capacity in both cell lines and patient samples. Critically, none of the identified NER deficient cell lines harbored mutations in any NER genes, suggesting that the prevalence of NER defects may currently be underestimated. Identification of compounds that induce the dNER gene expression signature led to the discovery that NER can be functionally impaired by GSK3 inhibition, leading to synergy when combined with cisplatin treatment. Furthermore, we predicted and validated multiple novel drugs that are synthetically lethal with NER defects using the dNER gene signature as a drug discovery platform. Taken together, our work provides a dynamic predictor of NER function that may be applied for therapeutic stratification as well as development of novel biological insights in human tumors.

Vaccination against COVID-19 is critical for immuno-compromised individuals, including patients with cancer. Systemic reactogenicity, a manifestation of the innate immune response to vaccines, occurs in up to 69% of patients following vaccination with RNA-based COVID-19 vaccines. Tumor regression can occur following an intense immune-inflammatory response and novel strategies to treat cancer rely on manipulating the host immune system. Here, we report spontaneous regression of metastatic salivary gland myoepithelial carcinoma in a patient who experienced grade 3 systemic reactogenicity, following vaccination with the mRNA-1273 COVID-19 vaccine. Histological and immunophenotypic inspection of the postvaccination lung biopsy specimens showed a massive inflammatory infiltrate with scant embedded tumor clusters (Insight: This case suggests that the mRNA-1273 COVID-19 vaccine stimulated anticancer immunity and tumor regression.

The AT-rich Interactive Domain 1A (ARID1A) is one of the most frequently mutated genes in gastric cancer. Here, we found that genetic variants in noncoding regions of ARID1A associated with altered protein levels by target sequencing. Notably, tumors with ARID1A variants in the 3’untranslated region (3’UTR) exhibited remarkably increased heterogeneity of ARID1A protein. In general, genetic variants and protein deficiency of ARID1A in tumors were associated with a better survival. Strikingly, altered patterns and heterogeneity of ARID1A protein expression were observed in peritumor tissues and carried significant implications in defining tumor immune contexture by multiplex immunohistochemistry. By analyzing the spatial distribution of TILs, we showed that reduced ARID1A protein levels in both tumor and peritumor tissues were significantly correlated with increased density and proximity of TILs to tumor cells. In contrast, high heterogeneity of ARID1A expression was associated with increased TIL density, but reduced proximity of TILs to tumor cells. Collectively, our study characterized ARID1A genetic alterations and its protein expression patterns in EOGC, demonstrating new strategies for clinically assessing its molecular impact on tumor onset and progression, tumor immune response, and patient survival.