Gourab Bhattacharjee

Selective depletion of plasma prekallikrein or coagulation factor XII inhibits thrombosis in mice without increased risk of bleeding

Recent studies indicate that the plasma contact system plays an important role in thrombosis, despite being dispensable for hemostasis. For example, mice deficient in coagulation factor XII (fXII) are protected from arterial thrombosis and cerebral ischemia-reperfusion injury. We demonstrate that selective reduction of prekallikrein (PKK), another member of the contact system, using antisense oligonucleotide (ASO) technology results in an antithrombotic phenotype in mice. The effects of PKK deficiency were compared with those of fXII deficiency produced by specific ASO-mediated reduction of fXII. Mice with reduced PKK had ∼ 3-fold higher plasma levels of fXII, and reduced levels of fXIIa-serpin complexes, consistent with fXII being a substrate for activated PKK in vivo. PKK or fXII deficiency reduced thrombus formation in both arterial and venous thrombosis models, without an apparent effect on hemostasis. The amount of reduction of PKK and fXII required to produce an antithrombotic effect differed between venous and arterial models, suggesting that these factors may regulate thrombus formation by distinct mechanisms. Our results support the concept that fXII and PKK play important and perhaps nonredundant roles in pathogenic thrombus propagation, and highlight a novel, specific and safe pharmaceutical approach to target these contact system proteases.

Inhibition of Vascular Permeability by Antisense-Mediated Inhibition of Plasma Kallikrein and Coagulation Factor 12

Hereditary angioedema (HAE) is a rare disorder characterized by recurrent, acute, and painful episodes of swelling involving multiple tissues. Deficiency or malfunction of the serine protease inhibitor C1 esterase inhibitor (C1-INH) results in HAE types 1 and 2, respectively, whereas mutations in coagulation factor 12 (f12) have been associated with HAE type 3. C1-INH is the primary inhibitor of multiple plasma cascade pathways known to be altered in HAE patients, including the complement, fibrinolytic, coagulation, and kinin-kallikrein pathways. We have selectively inhibited several components of both the kinin-kallikrein system and the coagulation cascades with potent and selective antisense oligonucleotides (ASOs) to investigate their relative contributions to vascular permeability. We have also developed ASO inhibitors of C1-INH and characterized their effects on vascular permeability in mice as an inducible model of HAE. Our studies demonstrate that ASO-mediated reduction in C1-INH plasma levels results in increased vascular permeability and that inhibition of proteases of the kinin-kallikrein system, either f12 or prekallikrein (PKK) reverse the effects of C1-INH depletion with similar effects on both basal and angiotensin converting enzyme (ACE) inhibitor-induced permeability. In contrast, inhibition of coagulation factors 11 (f11) or 7 (f7) had no effect. These results suggest that the vascular defects observed in C1-INH deficiency are dependent on the kinin-kallikrein system proteases f12 and PKK, and not mediated through the coagulation pathways. In addition, our results highlight a novel therapeutic modality that can potentially be employed prophylactically to prevent attacks in HAE patients.

Abstract LB-317: Potent in vivo pharmacology of AZD9150, a next-generation, constrained ethyl-modified antisense oligonucleotide targeting STAT3 in multiple preclinical cancer models.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Next generation sequencing technologies have greatly expanded our understanding of cancer genomes, epigenomes and transcriptomes. This knowledge, however, has not yet been effectively translated into improved cancer therapeutics, partly due to the inability of available therapeutic modalities to target the most promising cancer driver pathways. In contrast to other therapeutic approaches, the druggable universe is not limited with antisense technology as inhibitors can be rationally designed based on sequence information alone. Recent human clinical data has demonstrated potent activity of systemically-administered, unformulated, antisense oligonucleotides (ASOs) when targeted to liver expressed genes. However, robust activity in extra-heptatic tissues and tumors has been limited with existing ASO chemistries. Here we evaluate the activity of high affinity next generation (constrained ethyl) ASOs in extrahepatic tissues and tumors of multiple preclinical cancer models including spontaneous tumors, human tumor xenografts and several primary patient-derived xenograft models. As a test case we employed next generation ASOs to inhibit the difficult to drug transcription factor STAT3. ASOs targeting mouse STAT3 sequences and the human-specific STAT3 ASO (AZD9150) demonstrate potent and selective inhibition of target RNA and protein levels in tumors and tumor-associated stromal cells of a broad range of cancer models, resulting in strong antitumor activity in several models. These findings suggest that next generation ASO technology is now poised to become a key therapeutic modality to bridge the pharmacogenomic divide in cancer drug discovery. The STAT3 ASO inhibitor, AZD9150 is currently in human clinical studies including patients with lymphomas. Citation Format: Youngsoo Kim, Jeff Hsu, Tianyuan Zhou, Nancy Zhang, Richard Woessner, Murali VP Nadella, Deborah Lawson, Corinne Reimer, Guobin He, Joanna Schmidt, Xiaokun Xiao, Sarah Greenlee, Gourab Bhattacharjee, Gene Hung, Brett P. Monia, A. Robert MacLeod. Potent in vivo pharmacology of AZD9150, a next-generation, constrained ethyl-modified antisense oligonucleotide targeting STAT3 in multiple preclinical cancer models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-317. doi:10.1158/1538-7445.AM2013-LB-317

Abstract 2951: Potent antisense pharmacology of highly optimized antisense oligonucleotides in multiple transgenic, spontaneous and patient derived xenograft models of cancer reveals antitumor activity for the non-coding RNA MALAT-1.

Antisense technology holds great promise as a novel drug discovery platform that can rapidly translate discoveries from cancer genomics into highly selective therapeutics. Antisense oligonucleotices (ASOs) are particularly attractive, as they can be applied to difficult to drug target classes currently not tractable by other therapeutic modalities. Recent clinical demonstration of activity for ASOs in cancer patients supports the potential of this drug class, however, challenges in demonstrating robust antisense pharmacology in preclinical cancer models has slowed the progress of this technology as cancer therapeutics. Here we have employed a high resolution in-situ hybridization-based methodology (QuantigeneTM) among other detection methods, to demonstrate visually and quantitatively the activity of systemically administered, high potency next generation antisense oligonucleotides, in multiple preclinical cancer models. Cancer models evaluated include, transgenic models, chemically induced tumor, genetically predisposed mouse strains, cell line derived xenograft and patient derived xenograft models. As a test antisense target RNA sequence we chose the non-coding RNA MALAT1 (also called NEAT2) because it is ubiquitously expressed at high levels in most cell types and thus RNA levels could be readily visualize at the cellular level by Quantigene method. In addition, as MALAT-1 is overexpressed in many human tumors it also had the potential to be a therapeutically relevant target. We screened >1000 ASO sequences in vitro and identified highly potent mouse and mouse/human cross reactive MALAT-1 ASOs that reduced target RNA in cells in culture with IC50 values in the low nanomalor range (10-50 nM), without any lipid mediated delivery vehicles (ASO free uptake). Systemic delivery (s.c. administration of ASOs formulated in saline) of MALAT1 ASOs in vivo were well tolerated in all animals tested, and reduced target RNA by 70–>90% in the tumor cells of APC/min- mice, prostate tumor cells of the TRAMP model, DEN-induced HCC tumors as well as in the tumor cells of several human tumor xenograft models and in a patient derived NSCLC primary tumor explants model. Interestingly, MALAT-1 inhibition by ASOs was also associated with significant antitumor effects including inhibition of tumor formation and decreased BrdU positive cells in the polyps of APC/min- mice, decrease tumor growth in TRAMP prostate tumors and DEN HCC tumors and significant tumor growth delays in several xenograft and human tumor explant models. These data demonstrate unequivocal, potent, ASO mediated antisense activity of highly optimized next generation ASOs targeting MALAT-1 by systemic administration and highlight a previously uncharacterized role of the ncRNA MALAT1 as regulator of tumor growth in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2951. doi:1538-7445.AM2012-2951

Abstract B23: Selective inhibition of a long non-coding RNA (lncRNA), MALAT1 by antisense oligonucleotides results in significant anti-tumor effects in a variety of preclinical cancer models

An increasing body of evidence supports the notion that long noncoding RNAs (lncRNAs) play important roles in diseases including cancer. MALAT1 (also called NEAT2) was originally identified in tumors of highly metastatic non-small cell lung cancer (NSCLC), however, the functional role of MALAT1 in carcinogenesis has remained largely unknown. However, MALAT1 is highly expressed in various types of cancer and is implicated in the regulation of alternative splicing. To determine if MALAT1 constitutes an important driver of tumor growth in vivo, we developed potent antisense oligonucleotides (ASOs) that were able to achieve strong inhibition of MALAT1 in the tumor cells of a variety of preclinical models following systemic delivery. MALAT1 ASOs were well tolerated in rodents at doses leading to >95% inhibition of MALAT1 RNA in the liver. Inhibition of MALAT1 expression in tumor and tumor-associated stromal cells was determined by both q-RT-PCR using species-specific probe/primer sets and/or in situ ‘viewRNA’ technology, where target knockdown can be visualized on a cell by cell basis. Systemic administration of mouse MALAT1 ASOs resulted in a decrease in the numbers of polyps and proliferation index (BrdU (+)) in the small intestine of Apc min mouse model of colon cancer and correlated well with MALAT1 inhibition in the polyps. Mouse MALAT1 ASOs were also effective in a DEN-induced HCC model, where ASO treatment reduced the target RNA ~ 90% in tumor cells with a concomitant decrease in tumor numbers, while control ASO had no effects on either measure. In addition, higher expression of MALAT1 in non-treated tumors compared to adjacent normal hepatocytes was also clearly visualized by the in situ ‘view RNA’ method. Furthermore, MALAT1 ASO significantly delayed tumor growth in C26 colon cancer and reduced tumor size in TRAMP mouse model of prostate cancer, where the target RNA was decreased by 80% in tumor cells. In a human NSCLC patient-derived xenograft model, both significant MALAT1 RNA reduction and a delay in tumor growth were achieved after MALAT1 ASO treatment. The effects of MALAT1 downregulation by ASO were not limited to the inhibition of tumor growth alone. MALAT1 ASO treatment not only inhibited the growth of primary tumors in the EBC-1 human NSCLC xenograft model, but also resulted in a decrease in lung metastasis as measured by micro CT scanning. Furthermore, cross-species MALAT1 ASOs greatly improved the survival of animals bearing Hep3B human hepatocellular carcinoma (HCC) tumor orthotopically (48.5 days with control ASO vs 88 days with MALAT1 ASO, p=0.005). Taken together, these results demonstrate previously undiscovered roles of MALAT1 as an important regulator in vivo tumor growth and metastasis and suggest that selective inhibition of MALAT1 by ASO could have therapeutic value for the cancer treatment. Citation Format: Jeff Hsu, Guobin He, Gourab Bhattacharjee, Tianyuan Zhou, Chris May, Xiaokun Xiao, Gene Hung, Brett P. Monia, A. Robert MacLeod, Youngsoo Kim. Selective inhibition of a long non-coding RNA (lncRNA), MALAT1 by antisense oligonucleotides results in significant anti-tumor effects in a variety of preclinical cancer models [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr B23.

Abstract C136: Exploring the functional contribution of STAT3 activity in tumor and tumor-associated stromal cells with species-selective generation 2.5 antisense oligonucleotides in preclinical cancer models.

The transcription factor STAT3 is a point of convergence for multiple signaling pathways and is activated in both tumor and non-tumor stromal cells in the tumor microenvironment. STAT3 activation mediates the expression of genes involved in many aspect of tumorigenesis. Thus, STAT3 has been hotly pursued as a target for cancer. Although inhibitors of STAT3 upstream signaling pathways have shown anti-tumor activity in preclinical animal models, the relative contributions of STAT3 in tumor cells versus tumor-associated stromal cells to the observed antitumor effects remains unclear. To address this question we have developed potent next generation (Generation 2.5) antisense oligonucleotides (ASOs) that are selective for either mouse or human STAT3 and we haveassessed their specificity, potency, and antitumor activities in several preclinical cancer models. The safety of targeting STAT3 with ASOs was first confirmed in both rodents and non-human primates. STAT3 ASOs were very well tolerated at doses that resulted in complete abrogation of STAT3 expression in the liver of treated animals, Next, we evaluated the relative contribution of STAT3 in tumor cells and tumor-associated stromal cells in a human ovarian cancer xenograft model (SKOV3), using both mouse and human specific STAT3 ASOs. Mouse and human STAT3 ASOs selectively reduced their respective target STAT3 mRNA levels in SKOV3 tumors. However, when we evaluated the level of IL-6, a key cytokine in ovarian cancer and known to be regulated by STAT3, it was significantly reduced only by the inhibition of the STAT3 in the tumor associated-stromal cells (mouse STAT3). Moreover, the antitumor activity of the mouse STAT3 was superior to that observed with inhibition of STAT3 in the tumor cells themselves, suggesting that the tumor promoting effects of STAT3 in this model is mediated through STAT3 activation in tumor-associated stromal cells. To evaluate the effects of STAT3 inhibition in a mouse model of cancer we employed the APC min model in which mice develop intestinal neoplasia at a young age. Treatment of APC min mice with mouse STAT3 ASOs led to a >90% inhibition of STAT3 mRNA levels in both the small intestine and colon. In addition, STAT3 ASO treatment resulted in a significant decrease in the number of polyps in the intestine. Finally, we evaluated the effects of human-specific STAT3 ASO in NSCLC s.c. xenograft models using either PC9 or primary human tumor explants. Human STAT3 ASO treatment resulted in >50% inhibition in STAT3 mRNA levels in the tumor cells and resulted in significant tumor growth inhibition (∼50% and 90% >, respectively), suggesting that relative contributions of mouse stromal cells to tumor growth may vary in different tumor types. Taken together, these results demonstrate that STAT3 ASO can selectively downregulate STAT3 in both tumor and stromal compartments, affect anti-tumor activity in vivo and that stromal derived STAT3 activity is important contributor to tumor growth and survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C136.