Ashley T. Tsang

Outcomes of microwave ablation for colorectal cancer liver metastases: A single center experience

Surgical management of colorectal cancer liver metastases continues to evolve to optimize oncologic outcomes while maximizing parenchymal preservation. Long‐term data after intraoperative microwave ablation are limited. This study investigates outcomes and patterns of recurrence in patients who underwent intraoperative microwave ablation.

Thiosemicarbazones Functioning as Zinc Metallochaperones to Reactivate Mutant p53

Small-molecule restoration of wild-type structure and function to mutant p53 (so-called mutant reactivation) is a highly sought-after goal in cancer drug development. We previously discovered that small-molecule zinc chelators called zinc metallochaperones (ZMCs) reactivate mutant p53 by restoring zinc binding to zinc-deficient p53 mutants. The lead compound identified from the NCI-60 human tumor cell lines screen, NSC319726 (ZMC1), belongs to the thiosemicarbazone (TSC) class of metal ion chelators that bind iron, copper, magnesium, zinc, and other transition metals. Here, we have investigated the other TSCs, NSC319725 and NSC328784, identified in the same screen, as well as the more well studied TSC, 3-AP (Triapine), to determine whether they function as ZMCs. We measured the zinc Kd zinc ionophore activity, ability to restore zinc to purified p53 DNA binding domain (DBD), and ability to restore site-specific DNA binding to purified R175H-DBD in vitro. We tested all four TSCs in a number of cell-based assays to examine mutant p53 reactivation and the generation of reactive oxygen species (ROS). We found that NSC319725 and NSC328784 behave similarly to ZMC1 in both biophysical and cell-based assays and are heretofore named ZMC2 (NSC319725) and ZMC3 (NSC328784). 3-AP generates a ROS signal similar to ZMC1-3, but it fails to function as a ZMC both in vitro and in cells and ultimately does not reactivate p53. These findings indicate that not all TSCs function as ZMCs, and much of their activity can be predicted by their affinity for zinc.

U1 Adaptors suppress the KRAS-MYC oncogenic axis in human pancreatic cancer xenografts

Targeting KRAS and MYC has been a tremendous challenge in cancer drug development. Genetic studies in mouse models have validated the efficacy of silencing expression of both KRAS and MYC in mutant KRAS-driven tumors. We investigated the therapeutic potential of a new oligonucleotide-mediated gene silencing technology (U1 Adaptor) targeting KRAS and MYC in pancreatic cancer. Nanoparticles in complex with anti-KRAS U1 Adaptors (U1-KRAS) showed remarkable inhibition of KRAS in different human pancreatic cancer cell lines in vitro and in vivo. As a nanoparticle-free approach is far easier to develop into a drug, we refined the formulation of U1 Adaptors by conjugating them to tumor-targeting peptides (iRGD and cRGD). Peptides coupled to fluorescently tagged U1 Adaptors showed selective tumor localization in vivo. Efficacy experiments in pancreatic cancer xenograft models showed highly potent (>90%) antitumor activity of both iRGD and (cRGD)2-KRAS Adaptors. U1 Adaptors targeting MYC inhibited pancreatic cancer cell proliferation caused by apoptosis in vitro (40%–70%) and tumor regressions in vivo. Comparison of iRGD-conjugated U1 KRAS and U1 MYC Adaptors in vivo revealed a significantly greater degree of cleaved caspase-3 staining and decreased Ki67 staining as compared with controls. There was no significant difference in efficacy between the U1 KRAS and U1 MYC Adaptor groups. Our results validate the value in targeting both KRAS and MYC in pancreatic cancer therapeutics and provide evidence that the U1 Adaptor technology can be successfully translated using a nanoparticle-free delivery system to target two undruggable genes in cancer. Mol Cancer Ther; 16(8); 1445–55. ©2017 AACR.

Robotics and Pelvic Floor

Pelvic floor disorders can be categorized as primarily colorectal, gynecologic, or urologic. A multidisciplinary approach is often taken when there is a complex prolapse involving multiple organ systems [1]. Rectal prolapse, rectocele, enterocele, uterine prolapse, cystocele, and functional disorders of the pelvic floor muscles are all problems that can be treated surgically. Over a hundred operations have been described in the literature to repair pelvic organ prolapse. Generally, the operations can be divided into two broad categories, transabdominal and perineal. Evidence suggests that transabdominal procedures are more effective and applied to healthy patients while the perineal approach should be reserved for frail elderly patients with multiple comorbidities [2, 3].

262. Therapeutic Suppression of the KRAS-MYC Oncogenic Axis in Human Pancreatic Cancer Xenografts with U1 Adaptor Oligonucleotide / RGD Peptide Conjugates

U1 Adaptors are synthetic oligonucleotides that enable the U1 small nuclear ribonucleoprotein (U1 snRNP) complex to stably bind within the terminal exon of a specific pre-mRNA. This interferes with the obligatory polyadenylation step in mRNA maturation, causing selective destruction of the targeted mRNA inside the nucleus. In contrast to siRNA or antisense oligos, U1 Adaptors can accept extensive covalent modifications for nuclease resistance, targeted delivery or in-vivo imaging without loss of silencing activity, offering important advantages as therapeutic agents. A panel of candidate U1 Adaptors targeting human KRAS (KRAS Adaptors) was screened in vitro using the human pancreatic cancer cell line MIA-PaCa2. The best candidates reduced KRAS mRNA expression by up to 76% - as effectively as siRNA controls. Reduced KRAS protein expression was confirmed by western blot. Inhibition of cell growth in vitro and increased apoptosis were seen for both the MIA-PaCa2 (KRASG12C) and Panc1(KRASG12D) cell lines, but not in BxPC3, a KRASwildtype pancreatic cancer cell line. In a parallel project, U1 Adaptors targeting human MYC mRNA were designed and screened in B-cell lymphoma lines, where the best candidates reduced MYC mRNA levels by over 95%. Because of the observed relationship between activating KRAS mutation and MYC overexpression in pancreatic cancers, MYC Adaptors were tested in MIA-PaCa2 cells. MYC Adaptors also inhibited cell growth and increased apoptosis in vitro. U1 Adaptors were tested for efficacy in mice bearing subcutaneous MIA-PaCa2 xenograft tumors. For in-vivo delivery, Adaptor oligos were directly conjugated to a cyclic RGD-motif peptide (cRGD), which is a targeting ligand for specific integrin-family receptors overexpressed on parenchyma and endothelial cells of many solid tumors. Alternately, U1 Adaptor oligos were linked to “internalizing” RGD (iRGD), a variant RGD peptide that also triggers permeabilization of tumor endothelium and internalization by cells through secondary binding to neuropilin-1. KRAS Adaptors linked to cRGD or iRGD were administered by tail vein injections twice weekly for three to four weeks. Over a series of experiments, tumor growth was inhibited by averages of 68% to 93%. Tumor stasis or regression occurred in some treated mice. In a pilot study, MYC Adaptors conjugated to iRGD peptide were also highly effective in suppressing tumor growth and inducing tumor regression. Excised tumors were analyzed by qPCR and western blot, which confirmed reductions of the targeted mRNAs and proteins. We have shown that U1 Adaptors conjugated to tumor-targeting / tumor-penetrating peptides can effectively target human KRAS and MYC oncogenes in vivo. These results support the continued development of U1 Adaptor technology as a strategy for therapeutic suppression of KRAS, MYC and possibly other oncogene targets in pancreatic cancer.

Abstract 2097: Translating a mutant p53 reactivating drug (ZMC1) in murine pancreatic cancer models

Pancreatic cancer therapy suffers from a lack of effective chemotherapy. TP53 is second only to KRAS as the most commonly mutated gene in pancreatic cancer with point mutations occurring in 75% of patients. We identified ZMC1 as an allele specific mutant p53 reactivator and lead compound for mutant p53 targeted drug development. ZMC1 restores wildtype structure and function by functioning as a zinc metallochaperone to restore zinc binding to mutant p53 proteins with impaired zinc binding. Our aim was to translate this novel mechanism in vivo using murine pancreatic cancer models. We investigated the pharmacokinetics (PK) and pharmacodynamics (PD) of ZMC1 and performed efficacy studies for allele specificity in nude mice with subcutaneous tumors from murine pancreatic cancer cell lines derived from a genetically engineered mouse model (KPC) expressing mutant KrasG12D and different alleles of TP53 (WT, null, p53R172H, p53R270H). Tumor growth inhibition became apparent only in KPCp53-R172H xenografts but not in KPCp53-R270H. We then performed efficacy studies in the autochthonous KPC model and found that, ZMC1 extended the median survival from 15 to 26 days in the KPCp53-R172H mice (p = 0.05) but not in the KPCp53-R270H mice. We sought to improve the efficacy of ZMC1 by synthesizing it complexed with zinc (Zn-1) in a 2:1 molar ratio. Zn-1 significanlty increased the median survival of KPCp53-R172H mice from 15 to 35 days (p = 0.0042). The apoptosis rate in the tumors (by Immunohistochemistry staining with Cleaved Caspase 3) was also increased by treatment of ZMC1 and Zn-1. These studies indicate that ZMC1 can function in vivo as a mutant p53 targeted anti-cancer drug at doses that are well tolerated. Furthermore, ZMC1 can be optimized by synthesizing it complexed with zinc. Citation Format: Xin Yu, Ashley T. Tsang, Tracy Withers, John Gelleran, David Augeri, S. David Kimball, Darren R. Carpizo. Translating a mutant p53 reactivating drug (ZMC1) in murine pancreatic cancer models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2097.

Abstract 3833: Restoration of wildtype structure and function of mutant p53 by thiosemicarbozones using a novel zinc metallochaperone based mechanism

NSC319726 (ZMC1) is a small molecule that reactivates mutant p53 by restoration of WT structure and function to the most common p53 missense mutant (p53-R175H). We identified that ZMC1 functions as a zinc-metallochaperone, providing an optimal concentration of zinc to facilitate proper folding of p53 protein, and increasing cellular reactive oxygen species to transactivate the newly conformed p53-R175H (via post-translational modifications). ZMC1 was identified from an in silico screen of the NCI anti-cancer drug screen along with two other thiosemicarbazones (TSCs), NSC319725 and NSC328784. We investigated these TSCs to determine if they could reactivate mutant p53 using a zinc metallochaperone mechanism. We found that indeed these compounds could reactivate mutant p53 by functioning as zinc metallochaperones. In distinction, Triapine the only TSC in clinical development, does not function as a zinc metallochaperone and is not a mutant p53 reactivator. Citation Format: Xin Yu, Adam R. Blanden, Ashley T. Tsang, Saif Zaman, John Gelleran, David Augeri, S. David Kimball, Stewart N. Loh, Darren R. Carpizo. Restoration of wildtype structure and function of mutant p53 by thiosemicarbozones using a novel zinc metallochaperone based mechanism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3833.

694. Targeting KRAS in Pancreatic Cancer by Gene Silencing with U1 Adaptors

Activating mutations of the KRAS gene are key drivers of pancreatic cancer, but the KRAS protein has been refractory to small-molecule drugging. U1 Adaptors are oligonucleotides that enable the U1 small nuclear ribonucleoprotein complex to stably bind within the terminal exon of a specific pre-mRNA. This interferes with the obligatory polyadenylation step in mRNA maturation, causing selective destruction of the targeted mRNA in the nucleus. Unlike siRNA or antisense oligos, U1 Adaptors can be extensively modified for nuclease resistance or targeted delivery without loss of silencing activity, offering important advantages as therapeutic agents.We sought to translate U1 Adaptor technology to suppress KRAS in pancreatic cancer. A panel of U1 Adaptors targeting human KRAS (KRAS Adaptors) was screened in vitro using the human pancreatic cancer cell line MiaPaca-2 (KRAS G12D mutant). Candidate KRAS Adaptors reduced KRAS mRNA expression by up to 76%, as effectively as an siRNA control. Knockdown of KRAS protein expression was confirmed by western blot. Inhibition of cell growth in vitro was demonstrated for MiaPaca-2 and two additional pancreatic cancer cell lines, Panc1 (KRAS G12D mutant) and BXPC3 (KRAS wildtype).We evaluated KRAS Adaptors in mice bearing subcutaneous MiaPaca-2 xenograft tumors. For in-vivo delivery, the Adaptors were initially complexed with PAMAM dendrimers linked to a tumor-targeting, cyclic RGD peptide (cRGD), and administered by tail vein injection twice weekly for three weeks. Tumor growth was inhibited by as much as 68% compared to vehicle controls (p=0.0002). Excised tumors were analyzed by qPCR and IHC, which confirmed reductions of KRAS mRNA and protein.Although U1 Adaptors complexed with cRGD-dendrimers have been efficacious in this study and others, dendrimers have technical drawbacks and reported toxicities, and dendrimer-free formulations are preferable. As an alternative, we conjugated KRAS Adaptorsdirectly to cRGD peptide or to internalizing RGD (iRGD), which is a variant RGD peptide that triggers permeabilization of tumor endothelium and internalization by cells through secondary binding to neuropilin. The cRGD- and iRGD-conjugated KRAS Adaptors were tested for efficacy against subcutaneous MiaPaca-2 xenografts, and tumor growth was inhibited to equal or greater extent as with the original cRGD-dendrimer system. iRGD may be of particular benefit for pancreatic adenocarcinomas, which have a densely fibrotic stroma that impedes drug delivery. Enhanced delivery of small- and large-molecule therapeutics into primary pancreatic adenocarcinomas in KPC mice has been achieved previously by conjugating or coinjecting iRGD peptide.We have shown that U1 Adaptors can successfully target human KRAS both in vitro and in vivo. These results support the continued development of U1 Adaptor technology as a strategy for therapeutic suppression of KRAS in pancreatic cancer.

Abstract 1441: Adapting the Syngraft mouse model to study metastatic recurrence of pancreatic cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Pancreatic cancer is one of the most aggressive human malignancies with a 5-year survival rate for all stages of only 6%. While surgery remains the most effective treatment for pancreatic cancer, the majority of patients that undergo resection go on to die of pancreatic cancer recurrence. The biology of pancreatic recurrence is poorly understood due to the lack of sufficient mouse models to study this aspect of the disease. Our laboratory has adapted the Syngraft mouse model in order to study metastatic recurrence of pancreatic cancer. The Syngraft model is an orthotopic syngeneic mouse model using a pancreatic tumor cell line derived from the genetically engineered model (KrasG12D/+; Ink4a flox/flox) that has been modified with lentiviral vectors expressing mCherry and luciferase. After orthotopic injection, tumor formation was detected with the IVIS Spectrum using injected luciferin. Mice underwent distal pancreatectomy with spleenectomy when the primary pancreatic tumor measured ∼107 photons/sec/cm2, with a mean time to surgical resection at 19 days. Mice were then followed for metastatic recurrence to the liver using the IVIS Spectrum, with a mean time to recurrence of 23 days. We were able to establish a rate of metastatic recurrence in the Syngraft mouse model of 41% to the liver. Research is ongoing to enhance the metastatic efficiency of the injected cell lines and to further characterize the model for evidence of single cell and micrometastases in the liver. This model holds potential to be a useful tool to study the cell and molecular biology of pancreatic cancer recurrence. Citation Format: Crissy Dudgeon, Ashley Tsang, Neil Campbell, Eric Collisson, Darren Carpizo. Adapting the Syngraft mouse model to study metastatic recurrence of pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1441. doi:10.1158/1538-7445.AM2015-1441

Abstract B52: Therapeutic targeting of human KRAS in pancreatic cancer using a novel method of gene-silencing: U1 adaptors

Background: While genetic knockdown of RAS in mouse tumor models has substantiated it as a therapeutic target, there is no effective means of targeting RAS currently available in the clinic today. Numerous RNA interference-based studies targeting RAS have demonstrated therapeutic effects, however, effective delivery has been a major obstacle that has impeded this approach. U1 Adaptors are a novel technology for oligonucleotide-mediated gene silencing that act by selectively interfering with polyadenylation of messenger RNA (mRNA) inside the cell nucleus. Polyadenosine (PolyA) tail addition is an obligatory step in mRNA maturation and function, and its failure results in rapid degradation of the nascent message by endogenous nucleases. The eukaryotic U1 small nuclear ribonucleoprotein complex (U1 snRNP) is best known for its role as a pre-mRNA splicing factor, but also acts naturally to silence some genes by suppressing polyadenylation. U1 Adaptors are synthetic oligonucleotides that enable the U1 snRNP complex to stably bind to the terminal exon of any chosen pre-mRNA target, thereby interfering with polyA tail addition and causing it to be selectively degraded in the nucleus. The silencing mechanism of U1 Adaptors is distinct from those of siRNA or antisense oligonucleotides and this distinction confers an important advantage for their use as therapeutic agents. We have validated this technology in vivo demonstrating an 85% tumor growth inhibition by targeting BCL-2 and GRM-1 in human melanoma xenografts. Our in vivo proof-of-concept study relied on delivery of the U1 Adaptors non-covalently complexed with a nanoparticle comprised of a positively charged dendrimer covalently linked to a cyclic penta-peptide containing Arginine-Glycine-Aspartate referred to as the cRGD peptide, a widely-used tumor-targeting moiety. Methods/Results: We sought to translate the U1 Adaptor technology to target human KRAS. We first designed a set of U1 Adaptors for screening purposes targeting human KRAS at eight different positions along the human KRAS pre-mRNA located at the junction of the terminal exon (position 632) and untranslated region (UTR). We have evaluated these adaptors in vitro using the human pancreatic cancer cell line MIA Paca-2 (KRASG12D). Screening of these eight U1 Adaptors reveals a range of KRAS gene silencing as measured by quantitative PCR. Notably, Adaptors 2 and 3 silenced KRAS down to 27 and 24% respectively, as effective as the siRNA control. We then evaluated Adaptors 2 and 3 in human MIA Paca-2 xenografts. These adaptors were coupled to the cRGD-nanoparticle complex and administered by tail vein injection twice weekly. We observed significant tumor growth inhibition (37.3% by KRAS Adaptor 2, p=0.025, and 68.3% by KRAS Adaptor 3, p=0.0002, as compared to the vehicle control by Day 34. We also observed significant tumor growth inhibition with a U1 Adaptor targeting BCL-2 albeit to a lesser extent than KRAS Adaptor 3. Conclusion: We have demonstrated that the U1 Adaptor method of gene silencing can be successfully applied to target human KRAS both in vitro and in vivo. These results support the continued investigation of U1 Adaptor technology as a strategy for therapeutic targeting of RAS oncogenes. Citation Format: Ashley T. Tsang, Xin Yu, Rafal Goraczniak, Mark Brenneman, Samuel Gunderson, Darren R. Carpizo. Therapeutic targeting of human KRAS in pancreatic cancer using a novel method of gene-silencing: U1 adaptors. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B52. doi: 10.1158/1557-3125.RASONC14-B52