Extraordinary clinical response to ibrutinib in low-grade ovarian cancer guided by organoid drug testing.

Low-grade serous ovarian cancer (LGSOC) typically responds poorly to standard platinum-based chemotherapy and new therapeutic approaches are needed. We describe a remarkable response to targeted therapy in a patient with platinum-resistant, advanced LGSOC who had failed standard-of-care chemotherapy and two surgeries. The patient was in rapid decline and entering hospice care on home intravenous (i.v.) opioid analgesics and a malignant bowel obstruction requiring a G-tube. Genomic analysis of the patient's tumor did not indicate obvious therapeutic options. In contrast, a CLIA-certified drug sensitivity assay of an organoid culture derived from the patient's tumor identified several therapeutic choices, including Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, as well as the EGFR inhibitors afatinib and erlotinib. Following off-label administration of daily ibrutinib as monotherapy, the patient had an exceptional clinical turnaround over the following 65 weeks with normalization of CA-125 levels, resolution of the malignant bowel obstruction, halting of pain medications, and improvement of performance status from ECOG 3 to ECOG 1. After 65 weeks of stable disease, the patient's CA-125 levels began to rise, at which point the patient discontinued ibrutinib and began taking afatinib as monotherapy. The patient's CA-125 levels remained stable for an additional 38 weeks but due to anemia and rising CA-125 levels, the patient switched to erlotinib and is currently being monitored. This case highlights the clinical utility of ex vivo drug testing of patient-derived tumor organoids as a new functional precision medicine approach to identify effective personalized therapies for patients who have failed standard-of-care treatments.

Case report: ex vivo tumor organoid drug testing identifies therapeutic options for stage IV ovarian carcinoma.

Patients presenting with stage 4 ovarian carcinoma, including low-grade serous disease, have a poor prognosis. Although platinum-based therapies can offer some response, these therapies are associated with many side effects, and treatment resistance often develops. Toxic side effects along with disease progression render patients unable to receive additional lines of treatment and limit their options to hospice or palliative care. In this case report, we describe a patient with an unusual case of metastatic low-grade serous ovarian cancer with some features of high-grade disease who had received four previous lines of treatment and was suffering from atelectasis, pulmonary embolism, and hydronephrosis. A CLIA-certified drug sensitivity assay of an organoid culture derived from the patient's tumor (PARIS® test) identified several therapeutic options, including the combination of fulvestrant with everolimus. On this treatment regimen, the patient experienced 7 months of stable disease and survived nearly 11 months before succumbing to her disease. This case emphasizes the clinical utility of ex vivo drug testing as a new functional precision medicine approach to identify, in real-time, personalized treatment options for patients, especially those who are not benefiting from standard of care treatments.

Targeting BET Proteins BRD2 and BRD3 in Combination with PI3K-AKT Inhibition as a Therapeutic Strategy for Ovarian Clear Cell Carcinoma.

Ovarian clear cell carcinoma (OCCC) is a rare, chemo-resistant subtype of ovarian cancer. To identify novel therapeutic targets and combination therapies for OCCC, we subjected a set of patient-derived ovarian cancer cell lines to arrayed high-throughput siRNA and drug screening. The results indicated OCCC cells are vulnerable to knockdown of epigenetic gene targets such as bromodomain and extra-terminal domain (BET) proteins BRD2 and BRD3. Subsequent RNA interference assays, as well as BET inhibitor treatments, validated these BET proteins as potential therapeutic targets. Because development of resistance to single targeted agents is common, we next performed sensitizer drug screens to identify potential combination therapies with the BET inhibitor CPI0610. Several PI3K or AKT inhibitors were among the top drug combinations identified and subsequent work showed CPI0610 synergized with alpelisib or MK2206 by inducing p53-independent apoptosis. We further verified synergy between CPI0610 and PI3K-AKT pathway inhibitors alpelisib, MK2206, or ipatasertib in tumor organoids obtained directly from patients with OCCC. These findings indicate further preclinical evaluation of BET inhibitors, alone or in combination with PI3K-AKT inhibitors for OCCC, is warranted.

BET, SRC, and BCL2 family inhibitors are synergistic drug combinations with PARP inhibitors in ovarian cancer.

BACKGROUND: Homologous recombination deficiencies (HRD) are present in approximately half of epithelial ovarian cancers, for which PARP inhibitors (PARPi) are becoming a preferred treatment option. However, a considerable proportion of these carcinomas acquire resistance or harbour de novo resistance, posing a significant challenge to treatment. METHODS: To identify new combinatorial therapeutics to overcome resistance to PARPi, we employed high-throughput conditional RNAi and drug screening of patient-derived ovarian cancer cells. To prioritise clinically relevant drug combinations, we integrated empirical validation with analysis of The Cancer Genome Atlas (TCGA) and Genomics of Drug Sensitivity in Cancer (GDSC) datasets to nominate candidate targets and drugs, reaching three main findings. FINDINGS: Firstly, we found that the PARPi rucaparib enhanced the effect of BET inhibitors (CPI-203 & CPI-0610) irrespective of clinical subtype or HRD status. Additional drug combination screens identified that dasatinib, a non-receptor tyrosine kinase inhibitor, augmented the effects of rucaparib and BET inhibitors, proposing a potential broadly applicable triple-drug combination for high-grade serous and clear cell ovarian carcinomas. Secondly, rucaparib synergised with the BCL2 family inhibitor navitoclax, with preferential activity in ovarian carcinomas that harbour alterations in BRCA1/2, BARD1, or MSH2/6. Thirdly, we identified potentially antagonistic drug combinations between the PARPi rucaparib and vinca alkaloids, anthracyclines, and antimetabolites, cautioning their use in the clinic. INTERPRETATION: These findings propose therapeutic strategies to address PARP inhibitor resistance using agents that are already approved or are in clinical development, with the potential for rapid translation to benefit a broad population of ovarian cancer patients.

Functional Precision Medicine Identifies Novel Druggable Targets and Therapeutic Options in Head and Neck Cancer.

Purpose: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with high mortality and a lack of targeted therapies. To identify and prioritize druggable targets, we performed genome analysis together with genome-scale siRNA and oncology drug profiling using low-passage tumor cells derived from a patient with treatment-resistant HPV-negative HNSCC.Experimental Design: A tumor cell culture was established and subjected to whole-exome sequencing, RNA sequencing, comparative genome hybridization, and high-throughput phenotyping with a siRNA library covering the druggable genome and an oncology drug library. Secondary screens of candidate target genes were performed on the primary tumor cells and two nontumorigenic keratinocyte cell cultures for validation and to assess cancer specificity. siRNA screens of the kinome on two isogenic pairs of p53-mutated HNSCC cell lines were used to determine generalizability. Clinical utility was addressed by performing drug screens on two additional HNSCC cell cultures derived from patients enrolled in a clinical trial.Results: Many of the identified copy number aberrations and somatic mutations in the primary tumor were typical of HPV(-) HNSCC, but none pointed to obvious therapeutic choices. In contrast, siRNA profiling identified 391 candidate target genes, 35 of which were preferentially lethal to cancer cells, most of which were not genomically altered. Chemotherapies and targeted agents with strong tumor-specific activities corroborated the siRNA profiling results and included drugs that targeted the mitotic spindle, the proteasome, and G2-M kinases WEE1 and CHK1 We also show the feasibility of ex vivo drug profiling for patients enrolled in a clinical trial.Conclusions: High-throughput phenotyping with siRNA and drug libraries using patient-derived tumor cells prioritizes mutated driver genes and identifies novel drug targets not revealed by genomic profiling. Functional profiling is a promising adjunct to DNA sequencing for precision oncology. Clin Cancer Res; 24(12); 2828-43. ©2018 AACR.

MicroRNA miR-210 modulates cellular response to hypoxia through the MYC antagonist MNT.

The hypoxia-inducible factor (HIF) pathway is essential for cell survival under low oxygen and plays an important role in tumor cell homeostasis. We investigated the function of miR-210, the most prominent microRNA upregulated by hypoxia and a direct transcriptional target of HIFs. miR-210 expression was elevated in multiple cancer types and correlated with metastasis of breast and melanoma tumors. miR-210 overexpression in cancer cell lines bypassed hypoxia-induced cell cycle arrest and partially reversed the hypoxic gene expression signature. We identified MNT, a known MYC antagonist, as a miR-210 target. MNT mRNA contains multiple miR-210 binding sites in the 3' UTR and its knockdown phenocopied miR-210 overexpression. Furthermore, loss of MYC abolished miR-210-mediated override of hypoxia-induced cell cycle arrest. Comparison of miR-210 and MYC overexpression with MNT knockdown signatures also indicated that miR-210 triggered a "MYC-like" transcriptional response. Thus, miR-210 influences the hypoxia response in tumor cells through targeting a key transcriptional repressor of the MYC-MAX network.

Identification of SULF2 as a novel transcriptional target of p53 by use of integrated genomic analyses.

Microarray analysis has been useful for identifying the targets of many transcription factors. However, gene expression changes in response to transcription factor perturbation reveal both direct transcriptional targets and secondary gene regulation. By integrating RNA interference, gene expression profiling, and chromatin immunoprecipitation technologies, we identified a set of 32 direct transcriptional targets of the tumor suppressor p53. Of these 32 genes, 11 are not currently associated with the core p53 pathway. From among these novel pathway members, we focused on understanding the connection between p53 and SULF2, which encodes an extracellular heparan sulfate 6-O-endosulfatase that modulates the binding of growth factors to their cognate receptors and that has been shown to function as a tumor suppressor. Genetic and pharmacologic perturbation of p53 directly influences SULF2 expression, and similar to silencing of TP53, RNA interference-mediated suppression of SULF2 results in an impaired senescence response of cells to genotoxic stress. Thus, our integrated genomic approach has led to the identification of a novel mediator of p53 network biology.

A phosphorylated subpopulation of the histone variant macroH2A1 is excluded from the inactive X chromosome and enriched during mitosis.

Histone variants play an important role in numerous biological processes through changes in nucleosome structure and stability and possibly through mechanisms influenced by posttranslational modifications unique to a histone variant. The family of histone H2A variants includes members such as H2A.Z, the DNA damage-associated H2A.X, macroH2A (mH2A), and H2ABbd (Barr body-deficient). Here, we have undertaken the challenge to decipher the posttranslational modification-mediated "histone code" of mH2A, a variant generally associated with certain forms of condensed chromatin such as the inactive X chromosome in female mammals. By using female human cells as a source of mH2A, endogenous mH2A was purified and analyzed by mass spectrometry. Although mH2A is in low abundance compared with conventional histones, we identified a phosphorylation site, S137ph, which resides within the "hinge" region of mH2A. This lysine-rich hinge is an approximately 30-aa stretch between the H2A and macro domains, proposed to bind nucleic acids. A specific antibody to S137ph was raised; by using this reagent, S137 phosphorylation was found to be present in both male and female cells and on both splice variants of the mH2A1 gene. Although mH2A is generally enriched on the inactive X chromosome in female cells, mH2AS137ph is excluded from this heterochromatic structure. Thus, a phosphorylated subpopulation of mH2A appears to play a unique role in chromatin regulation beyond X inactivation. We provide evidence that S137ph is enriched in mitosis, suggestive of a role in the regulation of mH2A posttranslational modifications throughout the cell cycle.

MicroRNAs in the miR-106b family regulate p21/CDKN1A and promote cell cycle progression.

microRNAs in the miR-106b family are overexpressed in multiple tumor types and are correlated with the expression of genes that regulate the cell cycle. Consistent with these observations, miR-106b family gain of function promotes cell cycle progression, whereas loss of function reverses this phenotype. Microarray profiling uncovers multiple targets of the family, including the cyclin-dependent kinase inhibitor p21/CDKN1A. We show that p21 is a direct target of miR-106b and that its silencing plays a key role in miR-106b-induced cell cycle phenotypes. We also show that miR-106b overrides a doxorubicin-induced DNA damage checkpoint. Thus, miR-106b family members contribute to tumor cell proliferation in part by regulating cell cycle progression and by modulating checkpoint functions.

Long-distance combinatorial linkage between methylation and acetylation on histone H3 N termini.

Individual posttranslational modifications (PTMs) on histones have well established roles in certain biological processes, notably transcriptional programming. Recent genomewide studies describe patterns of covalent modifications, such as H3 methylation and acetylation at promoters of specific target genes, or "bivalent domains," in stem cells, suggestive of a possible combinatorial interplay between PTMs on the same histone. However, detection of long-range PTM associations is often problematic in antibody-based or traditional mass spectrometric-based analyses. Here, histone H3 from a ciliate model was analyzed as an enriched source of transcriptionally active chromatin. Using a recently developed mass spectrometric approach, combinatorial modification states on single, long N-terminal H3 fragments (residues 1-50) were determined. The entire modification status of intact N termini was obtained and indicated correlations between K4 methylation and H3 acetylation. In addition, K4 and K27 methylation were identified concurrently on one H3 species. This methodology is applicable to other histones and larger polypeptides and will likely be a valuable tool in understanding the roles of combinatorial patterns of PTMs.

Organismal differences in post-translational modifications in histones H3 and H4.

Post-translational modifications (PTMs) of histones play an important role in many cellular processes, notably gene regulation. Using a combination of mass spectrometric and immunobiochemical approaches, we show that the PTM profile of histone H3 differs significantly among the various model organisms examined. Unicellular eukaryotes, such as Saccharomyces cerevisiae (yeast) and Tetrahymena thermophila (Tet), for example, contain more activation than silencing marks as compared with mammalian cells (mouse and human), which are generally enriched in PTMs more often associated with gene silencing. Close examination reveals that many of the better-known modified lysines (Lys) can be either methylated or acetylated and that the overall modification patterns become more complex from unicellular eukaryotes to mammals. Additionally, novel species-specific H3 PTMs from wild-type asynchronously grown cells are also detected by mass spectrometry. Our results suggest that some PTMs are more conserved than previously thought, including H3K9me1 and H4K20me2 in yeast and H3K27me1, -me2, and -me3 in Tet. On histone H4, methylation at Lys-20 showed a similar pattern as H3 methylation at Lys-9, with mammals containing more methylation than the unicellular organisms. Additionally, modification profiles of H4 acetylation were very similar among the organisms examined.

Identification of histone H3 lysine 36 acetylation as a highly conserved histone modification.

Histone lysine acetylation is a major mechanism by which cells regulate the structure and function of chromatin, and new sites of acetylation continue to be discovered. Here we identify and characterize histone H3K36 acetylation (H3K36ac). By mass spectrometric analyses of H3 purified from Tetrahymena thermophila and Saccharomyces cerevisiae (yeast), we find that H3K36 can be acetylated or methylated. Using an antibody specific to H3K36ac, we show that this modification is conserved in mammals. In yeast, genome-wide ChIP-chip experiments show that H3K36ac is localized predominantly to the promoters of RNA polymerase II-transcribed genes, a pattern inversely related to that of H3K36 methylation. The pattern of H3K36ac localization is similar to that of other sites of H3 acetylation, including H3K9ac and H3K14ac. Using histone acetyltransferase complexes purified from yeast, we show that the Gcn5-containing SAGA complex that regulates transcription specifically acetylates H3K36 in vitro. Deletion of GCN5 completely abolishes H3K36ac in vivo. These data expand our knowledge of the genomic targets of Gcn5, show H3K36ac is highly conserved, and raise the intriguing possibility that the transition between H3K36ac and H3K36me acts as an "acetyl/methyl switch" governing chromatin function along transcription units.

Histone H2B deacetylation at lysine 11 is required for yeast apoptosis induced by phosphorylation of H2B at serine 10.

Chromatin alterations, induced by covalent histone modifications, mediate a wide range of DNA-templated processes, including apoptosis. Apoptotic chromatin condensation has been causally linked to the phosphorylation of histone H2B (serine 14 in human; serine 10 in yeast, H2BS10ph) in human and yeast cells. Here, we extend these studies by demonstrating a unidirectional, crosstalk pathway between H2BS10 phosphorylation and lysine 11 acetylation (H2BK11ac) in yeast. We demonstrate that the H2BK11 acetyl mark, which exists in growing yeast, is removed upon H(2)O(2) treatment but before H2BS10ph occurs, in a unidirectional fashion. H2B K11Q mutants are resistant to cell death elicited by H(2)O(2), while H2B K11R mutants that mimic deacetylation promote cell death. Our results suggest that Hos3 HDAC deacetylates H2BK11ac, which in turn mediates H2BS10ph by Ste20 kinase. Together, these studies underscore a concerted series of enzyme reactions governing histone modifications that promote a switch from cell proliferation to cell death.

Crossover homeostasis in yeast meiosis.

Crossovers produced by homologous recombination promote accurate chromosome segregation in meiosis and are controlled such that at least one forms per chromosome pair and multiple crossovers are widely spaced. Recombination initiates with an excess number of double-strand breaks made by Spo11 protein. Thus, crossover control involves a decision by which some breaks give crossovers while others follow a predominantly noncrossover pathway(s). To understand this decision, we examined recombination when breaks are reduced in yeast spo11 hypomorphs. We find that crossover levels tend to be maintained at the expense of noncrossovers and that genomic loci differ in expression of this "crossover homeostasis." These findings define a previously unsuspected manifestation of crossover control, i.e., that the crossover/noncrossover ratio can change to maintain crossovers. Our results distinguish between existing models of crossover control and support the hypothesis that an obligate crossover is a genetically programmed event tied to crossover interference.

Comprehensive phosphoprotein analysis of linker histone H1 from Tetrahymena thermophila.

Linker histone H1 is highly phosphorylated in normal growing Tetrahymena thermophila but becomes noticeably dephosphorylated in response to certain conditions such as prolonged starvation. Because phosphorylation of H1 has been associated with the regulation of gene expression, DNA repair, and other critical processes, we sought to use mass spectrometry-based approaches to obtain an in depth phosphorylation "signature" for this linker histone. Histone H1 from both growing and starved Tetrahymena was analyzed by nanoflow reversed-phase HPLC MS/MS following enzymatic digestions, propionic anhydride derivatization, and phosphopeptide enrichment via IMAC. We confirmed five phosphorylation sites identified previously and detected two novel sites of phosphorylation and two novel minor sites of acetylation. The sequential order of phosphorylation on H1 was deduced by using mass spectrometry to define the modified sites on phosphorylated H1 isoforms separated by cation-exchange chromatography. Relative levels of site-specific phosphorylation on H1 isolated from growing and starved Tetrahymena were obtained using a combination of stable isotopic labeling, IMAC, and tandem mass spectrometry.

Sterile 20 kinase phosphorylates histone H2B at serine 10 during hydrogen peroxide-induced apoptosis in S. cerevisiae.

Apoptosis is a highly coordinated cell suicide mechanism in vertebrates. Phosphorylation of serine 14 of histone H2B, catalyzed by Mst1 kinase, has been linked to chromatin compaction during apoptosis. We extend these results to unicellular eukaryotes by demonstrating that H2B is specifically phosphorylated at serine 10 (S10) in a hydrogen peroxide-induced cell death pathway in S. cerevisiae. H2B S10A mutants are resistant to cell death elicited by H(2)O(2) while H2B S10E phospho-site mimics promote cell death and induce the "constitutive" formation of condensed chromatin. Ste20 kinase, a yeast homolog of mammalian Mst1 kinase, translocates into the nucleus in a caspase-independent fashion and directly phosphorylates H2B at S10. Conservation of targeted H2B phosphorylation and the enzyme system responsible for the process point to an ancient mechanism of chromatin remodeling that likely plays an important role in governing cellular homeostasis in a wide range of organisms.

Identification of residues in yeast Spo11p critical for meiotic DNA double-strand break formation.

Saccharomyces cerevisiae Spo11 protein (Spo11p) is thought to generate the DNA double-strand breaks (DSBs) that initiate homologous recombination during meiosis. Spo11p is related to a subunit of archaebacterial topoisomerase VI and appears to cleave DNA through a topoisomerase-like transesterase mechanism. In this work, we used the crystal structure of a fragment of topoisomerase VI to model the Spo11p structure and to identify amino acid residues in yeast Spo11p potentially involved in DSB catalysis and/or DNA binding. These residues were mutated to determine which are critical for Spo11p function in vivo. Mutation of Glu-233 or Asp-288, which lie in a conserved structural motif called the Toprim domain, abolished meiotic recombination. These Toprim domain residues have been implicated in binding a metal ion cofactor in topoisomerases and bacterial primases, supporting the idea that DNA cleavage by Spo11p is Mg(2+) dependent. Mutations at an invariant arginine (Arg-131) within a second conserved structural motif known as the 5Y-CAP domain, as well as three other mutations (E235A, F260R, and D290A), caused marked changes in the DSB pattern at a recombination hotspot, suggesting that Spo11p contributes directly to the choice of DNA cleavage site. Finally, certain DSB-defective mutant alleles generated in this study conferred a semidominant negative phenotype but only when Spo11p activity was partially compromised by the presence of an epitope tag. These results are consistent with a multimeric structure for Spo11p in vivo but may also indicate that the amount of Spo11 protein is not a limiting factor for DSB formation in normal cells.