Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Development of a MUC16-Targeted Near-Infrared Antibody Probe for Fluorescence-Guided Surgery of Pancreatic Cancer

Pancreatic cancer (PDAC) is an extremely lethal disease with an overall survival rate of 10%. Surgery remains the only potentially curative treatment option, but resections are complicated by infiltrative disease, proximity of critical vasculature, peritumoral inflammation, and dense stroma. Surgeons are limited to tactile and visual cues to differentiate cancerous tissue from normal tissue. Furthermore, translating preoperative images to the intraoperative setting poses additional challenges for tumor detection, and can result in undetected and unresected lesions. Thus, PDAC has high rates of incomplete resections, and subsequently, disease recurrence. Fluorescence-guided surgery (FGS) has emerged as a method to improve intraoperative detection of cancer and ultimately improve surgical outcomes. Initial clinical trials have demonstrated feasibility of FGS for PDAC, but there are limited targeted probes under investigation for this disease, highlighting the need for development of additional novel biomarkers to reflect the PDAC heterogeneity. MUCIN16 (MUC16) is a glycoprotein that is overexpressed in 60-80% of PDAC, yet this biomarker has not been investigated for FGS of this disease. Therefore, the goal of this project was to develop a MUC16-targeted fluorescent probe for intraoperative identification of PDAC through optical surgical navigation. This dissertation describes the development of the fluorescent antibody conjugate, termed AR9.6-IRDye800, from inception to translational efficacy and safety studies. Initial studies demonstrated that AR9.6 bound to MUC16 in vitro, and demonstrated that binding was retained after conjugation to the near-infrared dye, IRDye800. Subcutaneous and orthotopic mouse models of pancreatic cancer demonstrated that this conjugate could target MUC16-expressing pancreatic cancer in vivo, and could identify PDAC intraoperatively, with significantly higher tumor to background ratios as compared to a non-specific IgG control. Metastatic lesions were identified under AR9.6-IRDye800 guidance, and fluorescence localization was observed microscopically in resected primary tumors and metastatic lesions. To build on the translational potential of this imaging probe, a humanized variant of the AR9.6 fluorescent conjugate was developed and investigated. This conjugate, termed huAR9.6-IRDye800, showed equivalent binding properties to its murine counterpart. Using an optimized dye:protein ratio of 1:1, in vivo studies demonstrated high tumor to background ratios in MUC16-expressing tumor models, and delineation of tumors in a patient-derived xenograft model. Safety, biodistribution, and toxicity studies were conducted, and demonstrated that huAR9.6-IRDye800 was safe, did not yield evidence of histological toxicity, and was well tolerated in vivo. The results from this work conclude that AR9.6-IRDye800 is an efficacious and safe imaging agent for identifying pancreatic cancer intraoperatively through fluorescence-guided surgery. Future studies will investigate additional large animal models, patient stratification, development of companion MUC16 diagnostics and theranostics, and further safety, toxicity and efficacy studies to enable clinical translation

The histone deacetylase inhibitor M344 as a multifaceted therapy for pancreatic cancer.

The histone deacetylase (HDAC) inhibitor vorinostat, used with gemcitabine and other therapies, has been effective in treatment of experimental models of pancreatic cancer. In this study, we demonstrated that M344, an HDAC inhibitor, is efficacious against pancreatic cancer in vitro and in vivo, alone or with gemcitabine. By 24 hours post-treatment, M344 augments the population of pancreatic cancer cells in G1, and at a later time point (48 hours) it increases apoptosis. M344 inhibits histone H3 deacetylation and slows pancreatic cancer cell proliferation better than vorinostat, and it does not decrease the viability of a non-malignant cell line more than vorinostat. M344 also elevates pancreatic cancer cell major histocompatibility complex (MHC) class I molecule expression, potentially increasing the susceptibility of pancreatic cancer cells to T cell lysis. Taken together, our findings support further investigation of M344 as a pancreatic cancer treatment

M344 decreases orthotopic pancreatic tumor growth when used as a treatment alone or in combination with gemcitabine.

(A) S2-013 cells were orthotopically implanted into the pancreas of female NU/J mice. After 8 days, the tumor volume for each mouse was monitored twice weekly with the VisualSonic Vevo 3100 Imaging System. At 15 days post-implantation of tumor cells, the mice were randomized into control or treatment groups with matched average tumor volumes. M344 was administered intraperitoneally at 10 mg/kg for 5 days per week (5 days on, 2 days off). Gemcitabine was given every 3 days intraperitoneally at 50 mg/kg. On Day 25 post-tumor implantation, the mice were euthanized and the tumors were resected and weighed. The changes in tumor volume over time are shown in (B) and representative images of tumors at 25 days post implantation are shown in (C). For statistical analysis, ordinary One-way ANOVA with Dunnett’s Multiple Comparisons test in GraphPad Prism Version 8.4.2 was used. The asterisks indicate the following p values: * p<0.05, ** p< 0.01, *** p<0.001.</p

Example of flow cytometry gating for the caspase 3/7 activation assay.

Details of the experimental process can be found in the legend for Fig 4. (TIF)</p

In pancreatic cancer cells, M344 causes cell cycle arrest in G₁.

Treatment of S2-013 cells with 1 or 10 μM M344 resulted in large increases in the populations accumulated in G1 at 24 hours (A), 48 hours (B), and 72 hours (C), as shown by propidium iodide staining and flow cytometry. Statistical comparisons were made using a Two-way ANOVA with Tukey’s Multiple Comparisons test in GraphPad Prism Version 8.4.2. The asterisks indicate the following p values: * p<0.05, ** p<0.01, ***p<0.001, **** p<0.0001.</p

M344-induced apoptosis is apparent by 48 hours and necrosis peaks at 72 hours.

S2-013 cells were treated with 0.1% DMSO control or M344 (1 μM or 10 μM) for 24, 48, or 72 hours. Caspase-3 and caspase-7 cleavage was simultaneously analyzed by using the CellEventTM Caspase-3/7 Green Flow Cytometry Assay Kit. The SYTOXTM AADvancedTM Dead Cell Stain included in the kit identified necrotic cells. Each error bar represents the standard error of the mean. Statistical comparisons of the results were done using a Two-way ANOVA with Tukey’s multiple comparisons test. The asterisks indicate the following p values: * <0.05, ** <0.01, *** <0.001, **** <0.0001.</p

M344 impairs viability in combination with gemcitabine.

S2-013 pancreatic cancer cells were treated with 0.1% DMSO, 10 μM M344, 100 nM gemcitabine, or 10 μM M344 + 100 nM gemcitabine. Viability was assessed by the trypan blue exclusion assay at 24, 48, and 72 hours and graphed as the number of live cells/total cells x 100. Each error bar represents the standard error of the mean. The results from 0.1% DMSO control treatment versus treatment with each M344 concentration were compared using Ordinary One-way ANOVA with Dunnett’s Multiple Comparisons test in GraphPad Prism Version 8.4.2. The asterisks indicate the p values: * p<0.05, ** p< 0.01, **** p<0.0001.</p

Immunoblotting of pancreatic cancer cell line HLA class I heavy chains following treatment of the cells with M344.

The immunoblot data displayed here correspond to Fig 9. The proteins were transferred after electrophoresis to a membrane that was then divided. The top portion was probed with anti-HSC 70 (loading control) antibody and the bottom portion was probed with the HC10 antibody (for HLA-B and–C heavy chains). The blots were imaged, and a long exposure and a short exposure are shown (on the left and right, respectively). (TIF)</p