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Background: Understanding the genetic basis for the molecular classification of Sinonasal Undifferentiated Carcinoma (SNUC) based on SMARCB1 may improve our understating regarding the nature of the disease. The objective of the study was to compare the genetic profile of SMARCB1-retained (SR-SNUC) and SMARCB1-deficient SNUC (SD-SNUC). Methods: Formalin-fixed, paraffin embedded tissue (FFPE) from treatment-naïve patients with SNUC were selected. Three cases of SR-SNUC, four cases of SD-SNUC, and four samples of non-tumor tissue (control samples) were selected. RNA sequencing was performed. Results: SR-SNUC had a higher number of variants (1 variant for every 15,000 bases) compared to SD-SNUC (1 variant every 29,000 bases). The ratio of missense to silent mutation ratio was higher for SR-SNUC (0.8) as compared to SD-SNUC (0.7). Approximately 1500 genes were differentially expressed between SR-SNUC and SD-SNUC. The genes that had a higher expression in SR-SNUC included TPD52L1, B3GNT3, GFY, TJP3, ELL3, CYP4F3, ALDH3B2, CKMT1B, VIPR1, SLC7A5, PPP2R2C, UPK3B, MUC1, ELF5, STY7 and H2AC14. The gene that had a higher expression in SD-SNUC was ZFHX4. Most of these genes were related to either protein translation or immune regulation. The most common (n=3, 75%) mechanisms of loss of SMARCB1 gene in SD-SNUC was loss of heterozygosity. Conclusion: RNA sequencing is a viable and informative approach for genomic profiling of archival SNUC samples. Both SMARCB1-retained and SD-SNUC were noted to have distinct genetic profiles underlying the molecular classification of these diseases.

Targeting cyclin-dependent kinases 4/6 (CDK4/6) represents a therapeutic option in combination with BRAF inhibitor and/or MEK inhibitor (MEKi) in melanoma; however, continuous dosing elicits toxicities in patients. Using quantitative and temporal in vivo reporting, we show that continuous MEKi with intermittent CDK4/6 inhibitor (CDK4/6i) led to more complete tumor responses versus other combination schedules. Nevertheless, some tumors acquired resistance that was associated with enhanced phosphorylation of ribosomal S6 protein. These data were supported by phospho-S6 staining of melanoma biopsies from patients treated with CDK4/6i plus targeted inhibitors. Enhanced phospho-S6 in resistant tumors provided a therapeutic window for the mTORC1/2 inhibitor AZD2014. Mechanistically, upregulation or mutation of NRAS was associated with resistance in in vivo models and patient samples, respectively, and mutant NRAS was sufficient to enhance resistance. This study utilizes an in vivo reporter model to optimize schedules and supports targeting mTORC1/2 to overcome MEKi plus CDK4/6i resistance.Significance: Mutant BRAF and NRAS melanomas acquire resistance to combined MEK and CDK4/6 inhibition via upregulation of mTOR pathway signaling. This resistance mechanism provides the preclinical basis to utilize mTORC1/2 inhibitors to improve MEKi plus CDK4/6i drug regimens. Cancer Discov; 8(5); 568–81. ©2018 AACR.See related commentary by Sullivan, p. 532.See related article by Romano et al., p. 556.This article is highlighted in the In This Issue feature, p. 517

Supplementary Table 4: Genes down-regulated (> 2-fold) by Stat5-shRNA and IST5-002 in K562

Supplementary Methods. Supplementary Figure 1. Chemical structures of the small molecule IST5-002 analogs. Supplementary Figure 2. IST5-002 inhibits phosphorylation of Stat5 in mouse and human breast cancer cells. Supplementary Figure 3. IST5-002 does not have significant inhibitory activity against 50 kinases tested. Supplementary Figure 4. IST5-002 does not affect phosphorylation of Pak1/2. Supplementary Figure 5. IST5-002 inhibits expression of Stat5 target genes in PC cells and in CML cells. Supplementary Figure 6. IST5-002 is not generally cytotoxic as determined by lack of cell death induction in cancer cell lines established from other organs. Supplementary Figure 7. CWR22Rv1 PC cells were inoculated subcutaneously into the flanks of castrated athymic nude mice supplied with sustained-release 5alpha-dihydrotestosterone (DHT)-pellets (n=10/group, 1 tumor/mouse, 1.5 ??107 CWR22Rv1 cells per site, 1 DHT pellet/mouse). Supplementary Figure 8. IST5-0-02 induces apoptosis in CWR22Rv1 tumors grown in nude mice and in clinical PCs cultured ex vivo in organ explant cultures. Supplementary Figure 9. IST5-002 induces extensive apoptotic death of imatinib-sensitive and -resistant CML cells.

Supplementary Table 3: Genes up-regulated (> 2-fold) by Stat5-shRNA and IST5-002 in K562

Supplementary Table 5: IST5-002 regulation of known Stat5 target genes in K562 cells.

Supplementary Table 2: Genes regulated by both Stat5-shRNA and IST5-002 in K562 cells, corresponding to the heatmap in Figure 2g.

Supplementary Figures

Materials and Methods

Supplemental Table 1: Structure-Activity-Relationship analysis.

Supplemental Table IMolecular and Clinical Characteristics of Primary AML Samples

RB loss occurs commonly in neoplasia but its contributions to advanced cancer have not been assessed directly. Here we show that RB loss in multiple murine models of cancer produces a prometastatic phenotype. Gene expression analyses showed that regulation of the cell motility receptor RHAMM by the RB/E2F pathway was critical for epithelial–mesenchymal transition, motility, and invasion by cancer cells. Genetic modulation or pharmacologic inhibition of RHAMM activity was sufficient and necessary for metastatic phenotypes induced by RB loss in prostate cancer. Mechanistic studies in this setting established that RHAMM stabilized F-actin polymerization by controlling ROCK signaling. Collectively, our findings show how RB loss drives metastatic capacity and highlight RHAMM as a candidate therapeutic target for treating advanced prostate cancer. Cancer Res; 77(4); 982–95. ©2016 AACR.

Supplemental Figure 2. RB deficiency promotes enhanced apoptotic pathway in response to ionizing radiation

PDF file - 464K, Mimicking tumor-derived BAF57 elevation in vitro.

Despite treatment with intensive chemotherapy, acute myelogenous leukemia (AML) remains an aggressive malignancy with a dismal outcome in most patients. We found that AML cells exhibit an unusually rapid accumulation of the repressive histone mark H3K27me3 on nascent DNA. In cell lines, primary cells and xenograft mouse models, inhibition of the H3K27 histone methyltransferase EZH2 to decondense the H3K27me3-marked chromatin of AML cells enhanced chromatin accessibility and chemotherapy-induced DNA damage, apoptosis, and leukemia suppression. These effects were further promoted when chromatin decondensation of AML cells was induced upon S-phase entry after release from a transient G1 arrest mediated by CDK4/6 inhibition. In the p53-null KG-1 and THP-1 AML cell lines, EZH2 inhibitor and doxorubicin cotreatment induced transcriptional reprogramming that was, in part, dependent on derepression of H3K27me3-marked gene promoters and led to increased expression of cell death–promoting and growth-inhibitory genes.In conclusion, decondensing H3K27me3-marked chromatin by EZH2 inhibition represents a promising approach to improve the efficacy of DNA-damaging cytotoxic agents in patients with AML. This strategy might allow for a lowering of chemotherapy doses, with a consequent reduction of treatment-related side effects in elderly patients with AML or those with significant comorbidities.Significance:Pharmacological inhibition of EZH2 renders DNA of AML cells more accessible to cytotoxic agents, facilitating leukemia suppression with reduced doses of chemotherapy.See related commentary by Adema and Colla, p. 359

Supplemental Table 2

nucleotide sequence

Detail description of microarray analysis and comparison with cancer stem cell dataset, and TMA of prostate cancer patients

Supplemental Figure 5. RB status is retained in biopsy proven local recurrence.

Purpose: BAF57, a component of the switching-defective and sucrose nonfermenting (SWI/SNF) chromatin-remodeling complex conglomerate, modulates androgen receptor activity to promote prostate cancer. However, the molecular consequences of tumor-associated BAF57 expression have remained undefined in advanced disease such as castration-resistant prostate cancer and/or metastasis.Experimental Design: Clinical human specimens of primary and metastatic prostate cancer were immunohistochemically examined for tumor-grade association of BAF57 expression. Global gene expression analyses were conducted in models mimicking tumor-associated BAF57 expression. Aberrant BAF57-dependent gene expression changes, bypass of androgen-mediated signaling, and chromatin-specific SWI/SNF complex alterations with respect to cytoskeletal remodelers such as integrins were validated. Cell migration assays were used to profile the biologic phenotypes conferred under conditions simulating tumor-derived BAF57 expression.Results: Immunohistochemical quantitation of primary human specimens revealed that BAF57 was significantly and aberrantly elevated as a function of tumor grade. Critically, gene expression analyses showed that BAF57 deregulation circumvented androgen-mediated signaling, elicited α2 integrin upregulation, and altered other SWI/SNF complex components at the α2 integrin locus. BAF57-dependent α2 integrin induction conferred a prometastatic migratory advantage, which was attenuated by anti-α2 integrin antibody blockade. Furthermore, BAF57 was found to be markedly upregulated in human prostate cancer metastases of the lung, lymph node, and dura.Conclusion: The findings herein, identifying tumor-associated BAF57 perturbation as a means to bypass androgen-signaling events that facilitate novel prometastatic phenotypes, link BAF57 upregulation to tumor dissemination. These data thereby establish BAF57 as a putative marker of metastatic potential that could be leveraged for therapeutic intervention. Clin Cancer Res; 19(10); 2657–67. ©2013 AACR.

PDF file - 130K

Supplemental data legends

Purpose: The retinoblastoma tumor suppressor (RB), a key regulator of cell-cycle progression and proliferation, is functionally suppressed in up to 50% of non–small cell lung cancer (NSCLC). RB function is exquisitely controlled by a series of proteins, including the CyclinD–CDK4/6 complex. In this study, we interrogated the capacity of a CDK4/6 inhibitor, palbociclib, to activate RB function.Experimental Design and Results: We employed multiple isogenic RB-proficient and -deficient NSCLC lines to interrogate the cytostatic and cytotoxic capacity of CDK 4/6 inhibition in vitro and in vivo. We demonstrate that while short-term exposure to palbociclib induces cellular senescence, prolonged exposure results in inhibition of tumor growth. Mechanistically, CDK 4/6 inhibition induces a proapoptotic transcriptional program through suppression of IAPs FOXM1 and Survivin, while simultaneously augmenting expression of SMAC and caspase-3 in an RB-dependent manner.Conclusions: This study uncovers a novel function of RB activation to induce cellular apoptosis through therapeutic administration of a palbociclib and provides a rationale for the clinical evaluation of CDK 4/6 inhibitors in the treatment of patients with NSCLC. Clin Cancer Res; 24(6); 1402–14. ©2018 AACR.

PDF file - 2249K, BAF57 aberrations govern pro-metastatic phenotypes.

in silico docking information

Supplemental Figure 1. Trop2 ICD abundance is maintained by Src kinase. Supplemental Figure 2. Canertinib, a tyrosine kinase inhibitor targeting EGFR reduces cell proliferation but does not affect Trop2 ICD abundance. Supplemental Figure 3. Schematic representation of the mechanism by which Src induction of cyclin D1 enhances cleavage of the membrane associated Trop2 complex to thereby enhance Trop2 ICD. Supplemental Figure 4. Human prostate cancer Tissue Microarray (TMA) IHC staining.

Supplemental Figure 1. RB Regulates RB/E2F Target Gene Transcription and Restricts NSCLC Tumor Burden. Supplemental Figure 2. RB Promotes Cellular Senescence in NSCLC. Supplemental Figure 3. RB Dependent In Vitro Cellular Apoptosis in Response to CDK4/6 Inhibition in NSCLC. Supplemental Figure 4. Mitochondrial Protein SMAC Regulates NSCLC Apoptosis In Vitro. Supplemental Figure 5. Human Transcriptome Array Identifies FOXM1 and Survivin as RB/E2F Signatures. Supplemental Figure 6. RB Mediates Non-small Cell Lung Cancer Apoptosis via RB/IAP/SMAC/Caspase 3 signaling axis.

Purpose: Perturbations in the retinoblastoma pathway are over-represented in advanced prostate cancer; retinoblastoma loss promotes bypass of first-line hormone therapy. Conversely, preliminary studies suggested that retinoblastoma-deficient tumors may become sensitized to a subset of DNA-damaging agents. Here, the molecular and in vivo consequence of retinoblastoma status was analyzed in models of clinical relevance.Experimental Design: Experimental work was performed with multiple isogenic prostate cancer cell lines (hormone sensitive: LNCaP and LAPC4 cells and hormone resistant C42, 22Rv1 cells; stable knockdown of retinoblastoma using shRNA). Multiple mechanisms were interrogated including cell cycle, apoptosis, and DNA damage repair. Transcriptome analysis was performed, validated, and mechanisms discerned. Cell survival was measured using clonogenic cell survival assay and in vivo analysis was performed in nude mice with human derived tumor xenografts.Results: Loss of retinoblastoma enhanced the radioresponsiveness of both hormone-sensitive and castrate-resistant prostate cancer. Hypersensitivity to ionizing radiation was not mediated by cell cycle or p53. Retinoblastoma loss led to alteration in DNA damage repair and activation of the NF-κB pathway and subsequent cellular apoptosis through PLK3. In vivo xenografts of retinoblastoma-deficient tumors exhibited diminished tumor mass, lower PSA kinetics, and decreased tumor growth after treatment with ionizing radiation (P < 0.05).Conclusions: Loss of retinoblastoma confers increased radiosensitivity in prostate cancer. This hypersensitization was mediated by alterations in apoptotic signaling. Combined, these not only provide insight into the molecular consequence of retinoblastoma loss, but also credential retinoblastoma status as a putative biomarker for predicting response to radiotherapy. Clin Cancer Res; 20(21); 5468–82. ©2014 AACR.

Supplemental Figure S1. RB loss promotes cell migration and invasion. Supplemental Figure S2. RB regulates RHAMM expression. Supplemental Figure S3. RB/E2F Binds on the RHAMM Promoter. Supplemental Figure S4. RHAMM overexpression drives cancer cell migration, invasion, and EMT. Supplemental Figure S5. RHAMM Inhibition Diminishes Migration and Invasion, but not Cellular Proliferation. Supplemental Figure S3. RB/E2F Binds on the RHAMM Promoter.

supplementary Table 1

Supplemental Figure 1. Trop2 ICD abundance is maintained by Src kinase. Supplemental Figure 2. Canertinib, a tyrosine kinase inhibitor targeting EGFR reduces cell proliferation but does not affect Trop2 ICD abundance. Supplemental Figure 3. Schematic representation of the mechanism by which Src induction of cyclin D1 enhances cleavage of the membrane associated Trop2 complex to thereby enhance Trop2 ICD. Supplemental Figure 4. Human prostate cancer Tissue Microarray (TMA) IHC staining.

PDF file - 2354K, Microarray analyses of BAF57 deregulation.

Supplemental Methods and Material

Supplemental Figure 1. RB status does not impinge on the alterations of cell cycle after radiation

Supplementary DataSupplemental Methods, Supplemental Figures and Legends

Supplementary Figure Legends Supplemental Figure S1. RB loss promotes cell migration and invasion. Supplemental Figure S2. RB regulates RHAMM expression. Supplemental Figure S3. RB/E2F Binds on the RHAMM Promoter. Supplemental Figure S4. RHAMM overexpression drives cancer cell migration, invasion, and EMT. Supplemental Figure S5. RHAMM Inhibition Diminishes Migration and Invasion, but not Cellular Proliferation.

Supplemental Figure 4. PLK3 expression promotes apoptotic cell death in the setting of RB loss.

PDF file - 681K, BAF57 dependent modulation of integrin signaling.

Supplemental Figure 3. NFκB is retained in the cytoplasm in the presence of an IκB dominant negative.

PDF file - 91K, Oligonucleotide Sequences and Kegg Pathway Summary.

Proteomic analysis of castration-resistant prostate cancer demonstrated the enrichment of Src tyrosine kinase activity in approximately 90% of patients. Src is known to induce cyclin D1, and a cyclin D1–regulated gene expression module predicts poor outcome in human prostate cancer. The tumor-associated calcium signal transducer 2 (TACSTD2/Trop2/M1S1) is enriched in the prostate, promoting prostate stem cell self-renewal upon proteolytic activation via a γ-secretase cleavage complex (PS1, PS2) and TACE (ADAM17), which releases the Trop2 intracellular domain (Trop2 ICD). Herein, v-Src transformation of primary murine prostate epithelial cells increased the proportion of prostate cancer stem cells as characterized by gene expression, epitope characteristics, and prostatosphere formation. Cyclin D1 was induced by v-Src, and Src kinase induction of Trop2 ICD nuclear accumulation required cyclin D1. Cyclin D1 induced abundance of the Trop2 proteolytic cleavage activation components (PS2, TACE) and restrained expression of the inhibitory component of the Trop2 proteolytic complex (Numb). Patients with prostate cancer with increased nuclear Trop2 ICD and cyclin D1, and reduced Numb, had reduced recurrence-free survival probability (HR = 4.35). Cyclin D1, therefore, serves as a transducer of v-Src–mediated induction of Trop2 ICD by enhancing abundance of the Trop2 proteolytic activation complex. Cancer Res; 76(22); 6723–34. ©2016 AACR.

PDF file - 554K, Metastatic loci from individual patients display differences in BAF57 elevation.