Carlo M. Contreras

Chemoproteomic Screening of Covalent Ligands Reveals UBA5 As a Novel Pancreatic Cancer Target

Chemical genetic screening of small-molecule libraries has been a promising strategy for discovering unique and novel therapeutic compounds. However, identifying the targets of lead molecules that arise from these screens has remained a major bottleneck in understanding the mechanism of action of these compounds. Here, we have coupled the screening of a cysteine-reactive fragment-based covalent ligand library with an isotopic tandem orthogonal proteolysis-enabled activity-based protein profiling (isoTOP-ABPP) chemoproteomic platform to rapidly couple the discovery of lead small molecules that impair pancreatic cancer pathogenicity with the identification of druggable hotspots for potential cancer therapy. Through this coupled approach, we have discovered a covalent ligand DKM 2-93 that impairs pancreatic cancer cell survival and in vivo tumor growth through covalently modifying the catalytic cysteine of the ubiquitin-like modifier activating enzyme 5 (UBA5), thereby inhibiting its activity as a protein that activates the ubiquitin-like protein UFM1 to UFMylate proteins. We show that UBA5 is a novel pancreatic cancer therapeutic target and show DKM 2-93 as a relatively selective lead inhibitor of UBA5. Our results underscore the utility of coupling the screening of covalent ligand libraries with isoTOP-ABPP platforms for mining the proteome for druggable hotspots for cancer therapy.

Comparative Proteomic Analysis of Whole-Gut Lavage Fluid and Pancreatic Juice Reveals a Less Invasive Method of Sampling Pancreatic Secretions

OBJECTIVES:There are currently no reliable, non-invasive screening tests for pancreatic ductal adenocarcinoma. The fluid secreted from the pancreatic ductal system (“pancreatic juice”) has been well-studied as a potential source of cancer biomarkers. However, it is invasive to collect. We recently observed that the proteomic profile of intestinal effluent from the bowel in response to administration of an oral bowel preparation solution (also known as whole-gut lavage fluid, WGLF) contains large amounts of pancreas-derived proteins. We therefore hypothesized that the proteomic profile is similar to that of pancreatic juice. In this study, we compared the proteomic profiles of 77 patients undergoing routine colonoscopy with the profiles of 19 samples of pure pancreatic juice collected during surgery.METHODS:WGLF was collected from patients undergoing routine colonoscopy, and pancreatic juice was collected from patients undergoing pancreatic surgery. Protein was isolated from both samples using an optimized method and analyzed by LC-MS/MS. Identified proteins were compared between samples and groups to determine similarity of the two fluids. We then compared our results with literature reports of pancreatic juice-based studies to determine similarity.RESULTS:We found 104 proteins in our pancreatic juice samples, of which 90% were also found in our WGLF samples. The majority (67%) of the total proteins found in the WGLF were common to pancreatic juice, with intestine-specific proteins making up a smaller proportion.CONCLUSIONS:WGLF and pancreatic juice appear to have similar proteomic profiles. This supports the notion that WGLF is a non-invasive, surrogate bio-fluid for pancreatic juice. Further studies are required to further elucidate its role in the diagnosis of pancreatic cancer.

Chronic Granulomatous Dermatitis Induced by Talimogene Laherparepvec Therapy of Melanoma Metastases

Talimogene laherparepvec (TVEC) is the first oncolytic viral immunotherapy approved by the FDA, for advanced melanoma consisting of genetically modified herpes simplex type 1 virus which selectively replicates causing tumor lysis, expressing granulocyte macrophage‐colony stimulating factor (GM‐CSF) and activating dendritic cells. Intratumoral injection of TVEC produces objective response in 41% of stage IIB‐IV M1a melanoma. However, clinical response assessment can be problematic due to immune‐related inflammation at established tumor sites. Herein, we report 5 cases of granulomatous dermatitis developing at sites of TVEC injection associated with pathologic complete response in 4 of 5 patients. Over 5 months, TVEC injections were administrated in a median of 20 tumors per patient for 9 median doses prior to biopsy of persistent, indurated nodules. Granulomatous dermatitis with melanophages and melanin pigment incontinence was observed in all samples without evidence of melanoma cells in 4 patients. The fifth patient was rendered melanoma‐free by resection of the 1 nodule out of 4 with persistent tumor. Repetitive administration of TVEC or other oncolytic viral immunotherapies mimicking unresolved infection can produce granulomatous inflammation confounding assessment of the degree of tumor response and need for additional TVEC therapy. Tumor biopsies are encouraged after 4 to 6 months of TVEC administration to differentiate melanoma from granulomatous inflammation. Patients with confirmed granulomatous dermatitis replace continued with remained in remission after treatment discontinuation. Inflammatory nodules typically regress spontaneously.

PRDM1 silences stem cell-related genes and inhibits proliferation of human colon tumor organoids

Significance Our previous studies demonstrated that PRDM1β is activated by p53 accumulation in human colorectal cancer cells. However, the function of PRDM1β in colorectal cancer cells and colon tumor organoids is not clear. Here we show that PRDM1β is a p53-response gene in human colon organoids and that low PRDM1 expression predicts poor survival in colon cancer patients. Also, PRDM1α and PRDM1β proteins repress a largely overlapping suite of genes, many of which are stem cell-related genes. Moreover, we show that forced expression of PRDM1β prevents the proliferation of colon tumor organoids. This work provides support for a role of PRDM1β in regulating normal colon stem cell proliferation. PRDM1 is a tumor suppressor that plays an important role in B and T cell lymphomas. Our previous studies demonstrated that PRDM1β is a p53-response gene in human colorectal cancer cells. However, the function of PRDM1β in colorectal cancer cells and colon tumor organoids is not clear. Here we show that PRDM1β is a p53-response gene in human colon organoids and that low PRDM1 expression predicts poor survival in colon cancer patients. We engineered PRDM1 knockouts and overexpression clones in RKO cells and characterized the PRDM1-dependent transcript landscapes, revealing that both the α and β transcript isoforms repress MYC-response genes and stem cell-related genes. Finally, we show that forced expression of PRDM1 in human colon cancer organoids prevents the formation and growth of colon tumor organoids in vitro. These results suggest that p53 may exert tumor-suppressive effects in part through a PRDM1-dependent silencing of stem cell genes, depleting the size of the normal intestinal stem cell compartment in response to DNA damage.

Abstract B10: Synthetic lethal screen to identify novel therapies targeting TP53-mutant colon cancer cells

We have performed a genome-wide synthetic lethal screen for pathways that are essential for the survival of colon cancer cells with TP53 mutations. RKO colon cancer cells are wild-type for TP53, and display robust p53-dependent growth arrest and apoptosis. We have utilized an isogenic pair of RKO cells differing only in TP53 gene status (Wild Type vs homozygous null derivative) to screen a genome-wide GeCKO CRISPR library and identified pathways that are novel therapeutic targets for TP53-mutant colon cancers. After infection of these cells with the GeCKO CRISPR library and one week of culturing in vitro, we performed deep GeCKO CRIPSR amplicon sequencing to enumerate and identify all CRISPRs that were underrepresented in TP53 mutant compared to wild type cells. We identified multiple CRISPRs targeting the CHEK1 and the SHH genes which were underrepresented in TP53 knockout cells. We then tested small molecule inhibitors of CHEK1 (UCN01) and the SHH receptor SMO (Cyclopamine) against isogenic pairs of RKO, HCT116, and DLD1 cells and confirmed that each molecule is significantly more toxic to cells with mutant TP53 than to those with wild-type TP53. Future studies will focus on testing these and additional targets alone and in combination against primary colon tumor and normal avatars, and preclinical models of TP53-mutant colon cancer. Citation Format: Charles C. Weige, Erin L. Anderson, Joseph Richardson, Carlo Contreras, Phillip J. Buckhaults. Synthetic lethal screen to identify novel therapies targeting TP53-mutant colon cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B10.

Abstract B14: TP53 synthetic lethal targets in colon cancer

We have performed a genome-wide screen for pathways that are synthetic lethal with TP53 mutations in colon cancer. RKO colon cancer cells are wild type for TP53, and display robust p53-dependent growth arrest and apoptosis. We utilized an isogenic pair of RKO cells differing only in TP53 gene status (wild type vs homozygous null derivative) to screen a genome-wide GeCKO CRISPR library. After infection of these cells with the library and culturing for one week in vitro, we performed deep amplicon sequencing to identify CRISPR guide RNAs that were underrepresented in TP53 knockout cells. Multiple CRISPR guide RNAs targeting the CHEK1 and SHH genes were underrepresented in TP53 knockout cells. To validate these targets, we used CRISPR genome engineering to create an independent panel of six RKO clones with biallelic disruption of TP53, and tested small molecule inhibitors of CHEK1 (UCN01) and the SHH receptor SMO (Cyclopamine). TP53 null cells were tenfold more sensitive to UCN01, and 100-fold more sensitive to Cyclopamine, compared to TP53 wild type cells. Finally, the relevance of these findings to human colon cancers was confirmed by the establishment and treatment of colon cancer organoid avatars with either wild type or mutant TP53. Normal colon organoids, and tumor organoids with wild type TP53 were unaffected by either drug, whereas TP53 mutant organoids were significantly growth inhibited. Future studies will focus on testing these and additional targets, alone and in combination, against primary colon tumor and normal avatars and other preclinical models of TP53-mutant colon cancer. Citation Format: Charles C. Weige, Changlong Liu, Erin L. Anderson, Carolyn E. Banister, Joseph Richardson, Carlo Contreras, Phillip J. Buckhaults. TP53 synthetic lethal targets in colon cancer. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr B14.

Abstract B4: Depletion of antibodies and serum-derived proteins to facilitate LC-MS detection of pancreas-specific proteins in pancreatic ductal secretions.

Pancreatic ductal secretions are an abundant source of proteins that may serve as biomarkers for the early detection of pancreatic adenocarcinoma. Most pancreatic ductal secretion proteomes published in the literature are dominated by serum proteins. Due to the invasive nature of procedures used to sample pancreatic secretions, it remains uncertain whether the serum proteins present in the samples are artifacts of blood introduced during sampling or true indicators of disease. In addition, serum proteins are not generally useful as specific disease markers and mask other less abundant pancreas-specific proteins of interest in the sample. The enrichment of pancreas-specific proteins in pancreatic ductal secretions increases the opportunity for uncovering specific biomarkers of pancreatic cancer. Pancreatic ductal secretions were collected from ten patients with pancreatic adenocarcinoma undergoing pancreatectomy during surgery according to IRB-approved protocols. Protease inhibitors were added and samples were stored at -80°C. Samples were extracted 3x with chloroform to remove hydrophobic contaminants followed by IgA depletion using SSL-7 agarose (Invivogen) and depletion with a Vivapure Anti-HSA human albumin depletion (Sartorius) or Seppro IgY 14 Spin Column (Sigma) serum protein depletion column. Depleted samples were precipitated with methanol/chloroform/water, dissolved into 2M urea with 50mM ammonium bicarbonate/ 10mM TCEP and digested with trypsin. The digest was processed on a C-18 column on an HPLC, dried, resuspended in 0.1% TFA and characterized by ESI-LC-MS/MS on an LTQ-Orbitrap. Data were searched using Mascot against the NCBI RefSeq database. The top 20 proteins in pancreatic secretions taken from pancreatic adenocarcinoma patients in order of abundance without depletion were albumin, transferrin, beta globin, alpha 2 globin, IgA-1 chain constant region, alpha-1 antitrypsin, complement component 3, carboxyl ester lipase, keratin 1, apolipoprotein A-1, haptoglobin, delta globin, Predicted protein similar to complement component C3, alpha-2 macroglobulin, pancreatic carboxypeptidase B1, IgA-2 chain constant region, apolipoprotein E, polymeric immunoglobulin receptor, IgG-1 constant region, and Ig lambda chains. Mascot scores for albumin were about 20 times higher than scores for the pancreas-specific proteins and about three times higher than scores for the other serum proteins. Twelve of the fourteen proteins which are depleted by the Seppro IgY 14 depletion columns are present as dominant proteins in most pancreatic secretion samples. Of the remaining top proteins, alpha-2, beta and delta globin are blood proteins which are not depleted. These are most likely the result of hemolyzed red blood cells during freezing. Carboxyl ester lipase, pancreatic carboxypeptidases A1, B1, elastase 3A and 3B, trypsin, regenerating islet-derived 1 alpha and beta, pancreatic lipase, chymotrypsin B1 and B2, pancreatitis-associated protein, pancreatic amylase alpha 2A and B, mesotrypsin, and phospholipase A2 are pancreas specific proteins which are detected with increased sensitivity once the interfering serum proteins are depleted. Data will be shown comparing the performance of the two columns in depletion efficiency as well as the effect of the addition of an additional depletion step of secretory IgA from the samples, which depleted IgA-1, IgA-2, polymeric immunoglobulin receptor, Ig-lambda and Ig-kappa chain proteins. In conclusion, IgA, albumin, and IgY 14 serum protein depletion improve the LC-MS analysis of pancreas-derived proteins in pancreatic secretions by reducing interference from abundant serum proteins. Citation Format: Jana D. Rocker, Carlo Contreras, Lee W. Thompson, Russell E. Brown, Jack A. DiPalma, Lewis K. Pannell. Depletion of antibodies and serum-derived proteins to facilitate LC-MS detection of pancreas-specific proteins in pancreatic ductal secretions. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B4.