Faculty Profile

Dr. Titto A. Augustine, Ph.D.

Titto A. Augustine, Ph.D.

Associate, Department of Medicine (Oncology)

Areas of Research: colorectal cancer, translational research, drug development, biomarker analysis, combinatorial therapy, immune checkpoint inhibition, small molecule PARP inhibitor, telomere length, oncolytic virus, reovirus

Professional Interests

My current research interests are,

1. Potentiating effect of reovirus administration in checkpoint immunotherapy in colorectal cancer

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in US. 70–80% of CRC is sporadic, and 20–30% has hereditary and 1–2% has inflammatory bowel disease components. About 84% of sporadic CRC has chromosomal instability (CIN), whereas as 13–16% has hypermutation and show microsatellite instability (MSI) due to defective DNA mismatch repair (MMR). The hypermutated MSI CRC is highly immunogenic because increasing amount of tumor-specific neoantigens and CD8-positive activated infiltrating cytotoxic T lymphocytes (CTLs) in the microenvironment. As these tumors express high level of immune checkpoint proteins, including PD-1, PD-L1, CTLA-4, and LAG-3 that inhibit CTLs from attacking tumors, immune checkpoint inhibitors (ICIs) have been successful in MSI CRC. Anti-PD-1 inhibitor works by releasing the programmed cell death protein 1 (PD-1) “brake” present on T cells. By preventing the PD-1 protein from engaging PD-L1, a protein present mainly on tumor cells, these immunotherapeutics suppress immune-inhibiting signals transmitted to T cells.

Metastatic CIN CRCs are non-hypermutated and microsatellite stable (MSS). MSS cancers tend to have higher proportion of KRAS oncogenic mutations (45-50%) than MSI counterparts (20-25%). Overall, ~30-50% of CRCs have mutated (abnormal) KRAS, indicating that up to 50% of patients might not respond to anti-epidermal growth factor receptor (EGFR) therapy. Reovirus (Reo) is a naturally occurring oncolytic double-stranded RNA virus. Earlier studies using tumor regression models in mice show that Reo, as part of replicating, preferentially exploits RAS/RAF/p38/MAPK signaling pathway and induces apoptosis in KRAS-mutated CRC cells. Reo is currently in clinical development to treat KRAS-mutated mCRC.

MSI CRC is highly sensitive to ICIs, whereas MSS is not. Sensitization of MSS CRC, the major subtype, for ICI will offer a tremendous improvement in the therapeutic options available. Research into immunotherapy is rapidly expanding, and clinical trials with current and in-development drugs are underway. In this project, we test the hypothesis that Reo attributes to innate and adaptive immune response and sensitizes MSS CRC to the inhibitory effects of anti-PD therapy, and thereby help about 96% of MSS mCRC patient population to respond positively to immunotherapy. We analyze to see if Reo infection, (1) induce the release of pro-inflammatory mediators and is regulated upon activation of T cells to express and secrete macrophage inflammatory protein-1α/β, granulocyte macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-α, IFN-γ, and tumor necrosis factor (TNF)-α, (2) suppress release of the immunosuppressive IL-10; and (3) increase activation and maturation of dendritic cells (DCs) and recruit effectors from both innate and adaptive immunity including CTLs and natural killer (NK) cells to facilitate tumor cell death. Role of Reo to release immune mediators in mitogen-activated protein kinase (MAPK)-, nuclear factor kappa B (NF-κB)- and protein kinase R (PKR)-dependent fashion, and to recruit DCs, CTLs and NK cells to altogether promote bystander immune-mediated cytotoxicity in MSS CRC microenvironment are unexplored areas. Using in vitro co-culture (cancer and immune cells), in vivo (mouse) allograft/syngeneic and our own novel unique KRAS mutant murine model, we propose to check this hypothesis through different angles. We’ll also perform molecular characterization studies and in-depth pathway analysis mainly to identify potential biomarkers of sensitivity for this combinatorial treatment. We believe that by sensitizing MSS mCRC for immunotherapy using Reo leads to revolutionary treatment modality.

2. Toll-like receptor 3 as a therapeutic target for reovirus infection in colorectal cancer

Reo, a naturally-occurring double stranded RNA virus, is in clinical development for patients with chemotherapy refractory KRAS-mutated tumors. The fact that more than 30-50% of human tumors harbor KRAS mutations had previously guided us to investigate the (cell killing) efficacy of Reo in KRAS-mutant CRC, and also to examine the cellular consequences of this biologic when used in combination with a chemotherapeutic drug called irinotecan, a topoisomerase I (enzyme participating in DNA replication) inhibitor and key component of first- and second-line treatments of metastatic CRC. Although much research was being undertaken to improve Reo’s efficacy by combining with other chemotherapeutic drugs, the plausible contribution of underlying signaling mechanisms that includes key proteins and immunological agents for virus recognition and dissemination largely remains unexplored. Toll-like receptor 3 (TLR3), a member of the  toll like receptor family of host innate immune system, is the pattern recognition motif/peptide protein present on the cell surface acceptable for double stranded RNA pathogens. We hypothesize that effective expression of host TLR3 dampens the infection potential of Reo by mounting a robust innate immune response.  Using TLR3 expressing commercial HEK-BlueTM-hTLR3 cells we confirm that TLR3 is the host pattern recognition motif responsible for detection of Reo. Knock down or deletion of TLR3, by utilizing molecular biology (siRNA) techniques, improves Reo’s infectivity which can be effectively harnessed towards better therapy for KRAS-mutated CRC. In order to explore further, we are seeking help of in vivo models of xenografts and an advanced specialized one of CRC, where tumors harbor mutations in KRAS grown locally/specific to the colon. Strategies to mitigate TLR3 response pathway may be utilized as a tool towards improved Reo efficacy to specifically target KRAS-mutated mCRC patients and to use in combination with other chemotherapeutic drugs.

3. Analysis of predictive biomarker for cetuximab and panitumumab sensitivity in colorectal cancer

According to ACS, an estimated 600,920 Americans will die from cancer in 2017. Among them, colorectal cancer (CRC) will account for over 50,260 deaths.  CRC is the third leading cause of cancer-related deaths in US when men and women are considered separately, and the second leading cause when both sexes are combined. The three primary treatment options for CRC are surgery, chemotherapy and radiation. Chemotherapeutic drugs include anti-EGFR (EGFR being cell-surface receptor that binds to extracellular molecule called epidermal growth factor (EGF) and helps for signaling and communication between cell and environment) monoclonal antibodies such as cetuximab, (Erbitux® by Bristol Myers Squibb; USFDA approval - 2004) and panitumumab (Vectibix® by Amgen Inc.; USFDA approval - 2007). Median overall survival for patients has improved to 24-30 months over the last decade primarily due to these drugs. These antibodies, which inactivate EGFR, are only effective in patients whose tumors lack KRAS mutation. The search for predictive biomarker has met partial success with incorporation of KRAS mutation as biomarker of exclusion of EGFR directed therapy.  Approximately 30-50% of CRCs are known to have a mutated KRAS, indicating that up to 50% of patients with CRC might respond to anti-EGFR antibody therapy. However, 40-60% of patients with wild-type KRAS tumors do not respond to such therapy. This wide discrepancy prompts us to search for a better predictive bio-marker of sensitivity for these drugs.

4. Telomere length as a novel predictive biomarker of sensitivity to anti-EGFR therapy in mCRC

We hypothesized that length of tandem repeats of short DNA sequence at the ends of chromosomes called ‘telomeres’ can act as a unique predictive bio-marker of sensitivity to anti-EGFR therapy. There is an association between EGFR/MAPK/PI3K pathways, length of telomeres (TL) and telomerase enzyme complex (that repairs telomeres so that they do not become progressively shorter during successive rounds of chromosomal replication/cell division) within clinical context. During the first part of study, we utilized an array of CRC cell lines, determined TL and sensitivity to cetuximab, which is a monoclonal antibody against EGFR, and identified TL as a biomarker of sensitivity. Later on, these data were validated in a robust multivariate model in patients treated with cetuximab and panitumumab. We found that, based on progression free survival (PFS), longer TL corresponded to better therapeutic outcome in patients with KRAS wild type mCRC. PFSs of other established therapeutic regimens for mCRC overlapped when separated into different cohorts based on TL. Our finding that longer TL portends for better outcome is therefore not an incidental finding, but indeed has biological plausibility. An added strength of this study was demonstration of hypothesis in vitro and among patient samples. TL didn’t appear to be a prognostic biomarker, but rather, a predictive one. This study, for first time, showed that malignant colonic epithelium corresponding to age-related decline in TL that is widely observed in nonmalignant cells such as peripheral blood leukocytes as well as colonocytes. The study also observed that when patients were categorized as either localized disease (stages 1–3), or stage 4 at diagnosis, there was a statistically significant difference in TL. Cancer tissue in general has lower TL than normal mucosa, and there appears to be a positive correlation between TL and telomerase. Therefore, telomerase, the enzyme constitutively expressed in cancer, is unable to increase TL in tumor cells beyond the adjoining normal mucosa. Our data can be used to incite large-scale analysis of TL from large registration studies and cooperative groups with access to patient’s DNA and clinical data. This can continue to unravel the mystery of the signaling pathways and the cell survival mechanisms that malignant tissues adapt as they progress. Further work was clearly focused on the ligand activated EGFR pathway, and downstream effectors, the MAPK and PI3K pathways, and study their roles in telomerase activity and subsequent effect on TL.

5. Targeting synthetic lethality by administering DNA damaging/cytotoxic agents and agents preventing DNA damage repair (PARP inhibitor)

Current standard of care for CRC consists of DNA interfering chemotherapeutic drugs such as oxaliplatin (Ox), irinotecan (I), 5-fluorouracil (5-FU), capecitabine etc. Synthetic lethality occurs when 2 cellular pathways involved in DNA repair are inhibited simultaneously, resulting in cell death, whereas inhibition of either pathway alone is not lethal. Poly (ADP-ribose) polymerase (PARP) is a modulator protein of DNA base excision repair, which constitutes a major mechanism for DNA damage repair and for genomic stability. Small molecule PARP inhibitors (PARPi) purportedly result in DNA damage and effective in killing tumors defective in additional DNA repair pathway(s). PARPi are promising agents in the treatment of cancer. CRC frequently harbors defects in DNA repair pathways, leading to genomic instability. MSI, caused by the loss of MMR, another form of DNA damage repair, is detected in about 15% of all CRC. In this study, we tried to explore best combinatorial treatment for synthetic lethality by combining an array of DNA damaging chemotherapeutic agents and PARPis in presence and absence of MMR status, MSS and MSI, respectively. The DNA damaging agents used in the study are Ox, I, and 5-FU, and PARPis are veliparib (V), rucaparib (R), olaparib (Ol) and PJ34. While combinations of Ox and PJ34, and, Ox and V showed antagonistic effect, combination of cytotoxic drug, I, and PARPi, R, proved to be eliciting best synergy of anticancer effect in CRC cell lines and animal (mouse) model. Our results indicate that the inhibitory or anticancer effect of I and R is not dependent on particular sequence of drug administration, but mediated via certain genes participating in cell cycle arrest and apoptosis, a form of cell death. Synergistic effect of I and R is confirmed through xenografted animal models generated in athymic nude mice, which were immunodeficient.

Selected Publications

  1. Toll like receptor 3 as an immunotherapeutic target for KRAS mutated colorectal cancer. Maitra R, Augustine T, Dayan Y, Chandy C, Coffey M, Goel S. Oncotarget. 2017 Apr 4. doi: 10.18632/oncotarget.16812. [Epub ahead of print]
  2. Telomere length is a novel predictive biomarker of sensitivity to anti-EGFR therapy in metastatic colorectal cancer. Augustine TA, Baig M, Sood A, Budagov T, Atzmon G, Mariadason JM, Aparo S, Maitra R, Goel S. Br J Cancer. 2015 Jan 20;112(2):313-8. doi: 10.1038/bjc.2014.561. Epub 2014 Nov 20.
  3. Oncolytic reovirus preferentially induces apoptosis in KRAS mutant colorectal cancer cells, and synergizes with irinotecan. Maitra R, Seetharam R, Tesfa L, Augustine TA, Klampfer L, Coffey MC, Mariadason JM, Goel S. Oncotarget. 2014 May 15;5(9):2807-19.
  4. Potent antitumor and antineoplastic efficacy of baicalein on benzo(a)pyrene-induced experimental pulmonary tumorigenesis. Naveenkumar C, Asokkumar S, Raghunandhakumar S, Jagan S, Anandakumar P, Augustine TA, Kamaraj S, Devaki T. Fundam Clin Pharmacol. 2012 Apr;26(2):259-70. doi: 10.1111/j.1472-8206.2010.00910.x. Epub 2011 Feb 16.
  5. Silymarin inhibited proliferation and induced apoptosis in hepatic cancer cells. Ramakrishnan G, Lo Muzio L, Elinos-Báez CM, Jagan S, Augustine TA, Kamaraj S, Anandakumar P, Devaki T. Cell Prolif. 2009 Apr;42(2):229-40. doi: 10.1111/j.1365-2184.2008.00581.x.
  6. Silymarin downregulates COX-2 expression and attenuates hyperlipidemia during NDEA-induced rat hepatocellular carcinoma. Ramakrishnan G, Elinos-Báez CM, Jagan S, Augustine TA, Kamaraj S, Anandakumar P, Devaki T. Mol Cell Biochem. 2008 Jun;313(1-2):53-61. doi: 10.1007/s11010-008-9741-5. Epub 2008 Mar 31.
  7. Effect of silymarin on N-nitrosodiethylamine induced hepatocarcinogenesis in rats. Ramakrishnan G, Augustine TA, Jagan S, Vinodhkumar R, Devaki T. Exp Oncol. 2007 Mar;29(1):39-44.

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Albert Einstein College of Medicine
Jack and Pearl Resnick Campus
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