Colorectal cancer (CRC) is the third most commonly diagnosed cancer, and the second leading cause of cancer-related death in the 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 neoantigens and CD8-positive activated infiltrating cytotoxic T lymphocytes (CTLs) in the tumor microenvironment (TME). However, the high expression of checkpoint molecules PD-1, PD-L1, CTLA-4, LAG-3, and IDO in MSI CRC distinguishes them from microsatellite stable (MSS) CRC and creates an immunosuppressive TME that may help evading immune destruction by CTLs.

Traditional means of management of CRC is based on the type and stage including surgery, radiation, chemotherapy, targeted therapy and immunotherapy. Therapies aimed at overcoming the mechanisms of peripheral tolerance, in particular by blocking the inhibitory checkpoints, offer the potential to generate antitumor activity, either as monotherapies or in synergism with other therapies that directly or indirectly enhance presentation of tumor epitopes to the immune system. Our group focuses on developing single agent and combinatorial treatment regimens and analyzing biomarkers that predict the response.

1. Potentiating effect of immune checkpoint blockade on poly [ADP-ribose] polymerase inhibitor mediated cytotoxicity in CRC

There are two main types of targeted therapies: small molecule medicines and monoclonal antibodies. Small molecule inhibitors targeting DNA damage repair pathways have been particularly of interest in CRC. Small molecule inhibitors of poly [ADP-ribose] polymerase-1 (PARP-1) have been shown to have significant clinical potential and third generation PARP inhibitors (PARPis) are currently being investigated in CRC clinical trials. PARP-1 is primarily involved in base excision repair, although its role in regulating other repair pathways including homologous recombination (HR), MMR etc. is noteworthy. PARP inhibition leads to defects in PAR metabolism that results in increased DNA damage and are deleterious to cells. Immune checkpoints refer to a plethora of inhibitory pathways hardwired into the immune system that are crucial for maintaining self-tolerance and modulating the duration and amplitude of physiological immune responses in peripheral tissues in order to minimize collateral tissue damage. Immune checkpoint inhibitors (ICIs), such as anti-PD-1 or anti-PD-L1, and PARPis have had substantial impact on treatment outcomes in patients with wide range of solid tumor malignancies. Non-clinical data have demonstrated a direct association between DNA damage and immune responses, which supports the combined use of ICIs and PARPis. PARPis upregulate PD-L1, primarily via glycogen synthase kinase-3β inactivation and, in the absence of an ICI, can result in cancer cells that have increased resistance to CTL-mediated cell death. Additionally, tumors with BRCA mutations or HR deficiency have been associated with high neoantigen load, improved CD8 cell to CD4 cell ratio, increased peritumoral T cells, and upregulated PD-L1 expression. The blockade of the PD-1 – PD-L1 axis with an ICI might potentiate PARPi-induced tumor suppression. 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. The efficacy of PD-1 pathway blockade also correlates with DNA repair pathway mutations and neoantigen burden. A CRC tumor that responds to PARPi might have enhanced sensitivity to combined treatment with a PARPi and an anti-PD-1 antibody, and the efficacy and safety of the two drugs administered together deserves further investigation.

We are poised to utilize state-of-the-art technology - CRISPR-Cas9 screening – to establish potential biomarkers in relation to DNA damage repair, and a remarkable in vivo model - humanized NSG-SGM3 triple transgenic mouse that hosts patient-derived xenograft - to validate the findings. We analyze PARPi’s property to induce immunogenic cell death (ICD) – a form of cell death that elicits immune response or even circumvent immune tolerance against dying cancer cells - via brining a tumor vaccination model into light and examining the role of damage‐associated molecular patterns (DAMPs). ICD is marked by chronic exposure of DAMPs in the TME which stimulates dendritic cell recruitment and activation and production of a dysfunctional or antitumor [adaptive] immune system. Induction of ICD can improve the clinical outcomes of patients with certain malignancies after chemotherapy. Ability of a specific stimulus to cause bona fide ICD can be measured using surrogate markers such as cell surface-exposed calreticulin and heat shock proteins (HSP70 and HSP90), and extracellular ATP and high mobility group box 1 (HMGB1), and/or the processes that underlie their emission, such as endoplasmic reticulum stress/reactive oxygen species, autophagy and necrotic plasma membrane permeabilization. Our group will study the mechanisms related to DAMP regulation and associated pathways that connects them with pattern recognition receptors (PPR) on the surface of immune cells.

2. Combinatorial efficacy of oncolytic reovirus and immunotherapy in MSS subtype of CRC

Over the past 5 years, immune based therapy has found its place in the forefront of the “war on cancer”. The determination of KRAS, BRAF, and MMR status (MSI/MSS) has become an indispensable tool in therapeutic planning of CRC patients. Only about 3-4% of metastatic CRC (mCRC) is MSI. CIN mCRCs are non-hypermutated and 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. Discovering ways of rendering this vast subset of tumors to the beneficial effects of immune therapy is a formidable and critical goal. Reovirus (Reo) is a naturally occurring oncolytic double-stranded RNA (dsRNA) 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 (CRC cell lines and immune cells), in vivo (mouse) allograft/syngeneic and our own novel and 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.

3. Toll-like receptor 3 as a therapeutic target for reo infection in CRC

Reo 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 mCRC. 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 PRR protein present on the cell surface acceptable for dsRNA 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 RNAi 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.

4. Analysis of predictive biomarker for anti-EGFR treatment in CRC

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.

5. 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 and identified TL as a biomarker of sensitivity. Later on, the data was 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 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.

6. Targeting synthetic lethality by administering a chemotherapeutic drug and PARPi in CRC

Standard of care for CRC consists of DNA interfering/cytotoxic or chemotherapeutic drugs such as oxaliplatin (Ox), irinotecan (I), 5-fluorouracil (5-FU) and capecitabine. 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. PARPis purportedly result in DNA damage and effective in killing tumors defective in additional DNA repair pathway(s). CRC frequently harbors defects in DNA repair pathways, leading to genomic instability, particularly in MSI condition. 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 statuses, 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 indicated 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, particularly S phase, and early-stage apoptosis, a form of programmed cell death. Synergistic effect of I and R was confirmed through xenografted animal models generated in athymic nude (immunodeficient) mice. Further molecular characterization of key players of mechanistic pathways needed to be explored.

1.           Jiffry J, Thavornwatanayong T, Rao D, Fogel EJ, Saytoo D, Nahata R, Guzik H, Chaudhary I, Augustine T, Goel S, Maitra R. Oncolytic reovirus (pelareorep) induces autophagy in KRAS-mutated colorectal cancer. Clin Cancer Res. 2020 Nov 9:clincanres.2385.2020. doi: 10.1158/1078-0432.CCR-20-2385.

2.           Parakrama R, Fogel E, Chandy C, Augustine T, Coffey M, Tesfa L, Goel S, Maitra R. Immune characterization of metastatic colorectal cancer patients post reovirus administration. BMC Cancer. 2020 Jun 18;20(1):569. doi: 10.1186/s12885-020-07038-2. PMID: 32552875; PMCID: PMC7301987.

3.           Rao D, Parakrama R, Augustine T, Liu Q, Goel S, Maitra R. Immunotherapeutic advances in gastrointestinal malignancies. NPJ Precis Oncol. 2019;3:4. Published 2019 Feb 5. doi:10.1038/s41698-018-0076-8.

4.           Maitra R, Thavornwatanayong T, Venkatesh MK, Chandy C, Vachss D, Augustine T, Guzik H, Koba W, Liu Q, Goel S. Development and Characterization of a Genetic Mouse Model of KRAS Mutated Colorectal Cancer. Int J Mol Sci. 2019;20(22):5677. Published 2019 Nov 13. doi:10.3390/ijms20225677.

5.           Augustine T, Maitra R, Zhang J, Nayak J, Goel S. Sensitization of colorectal cancer to irinotecan therapy by PARP inhibitor rucaparib. Invest New Drugs. 2019;37(5):948–960. doi:10.1007/s10637-018-00717-9.

6.           Rao D, Mallick AB, Augustine T, Daroqui C, Jiffry J, Merla A, Chaudhary I, Seetharam R, Sood A, Gajavelli S, Aparo S, Rajdev L, Kaubisch A, Chuy J, Negassa A, Mariadason JM, Maitra R, Goel S. Excision repair cross-complementing group-1 (ERCC1) induction kinetics and polymorphism are markers of inferior outcome in patients with colorectal cancer treated with oxaliplatin. Oncotarget. 2019;10(53):5510–5522. Published 2019 Sep 17. doi:10.18632/oncotarget.27140

7.           Augustine T, Maitra R, Goel S. Telomere length regulation through epidermal growth factor receptor signaling in cancer. Genes and Cancer. 2017 May;8(5-6):550-558. PubMed PMID: 28740573.

8.           Maitra R, Augustine T, Dayan Y, Chandy C, Coffey M, Goel S. Toll like receptor 3 as an immunotherapeutic target for KRAS mutated colorectal cancer. Oncotarget. 2017 May;8(21):35138-35153. PubMed PMID: 28422714.

9.           Maitra R, Augustine TA, Venkatesh MK, Goel S. Reovirus Oncolysis- Role of cyclin-dependent kinase inhibitor 1. Cancer Cell & Microenvironment. 2015; 2: e750. DOI: 10.14800/ccm.750.

10.        Augustine TA, Baig M, Sood A, Budagov T, Atzmon G, Mariadason JM, Aparo S, Maitra R, Goel S. Telomere length is a novel predictive biomarker of sensitivity to anti-EGFR therapy in metastatic colorectal cancer. Br J Cancer. 2015 Jan;112(2):313-8. PubMed PMID: 25412235

11.        Maitra R, Seetharam R, Tesfa L, Augustine TA, Klampfer L, Coffey MC, Mariadason JM, Goel S. Oncolytic reovirus preferentially induces apoptosis in KRAS mutant colorectal cancer cells, and synergizes with irinotecan. Oncotarget. 2014 May;5(9):2807-19. PubMed PMID: 24798549; PubMed PMCID: PMC4058046.

12.        Maitra R, Nayak JB, Merla A, Sood A, Augustine TA, Baig M, Reynolds DM, Mariadason JM, Aparo S, and Goel S. A Single Nucleotide Polymorphism in the MGMT gene is a novel prognostic biomarker in patients with metastatic colorectal cancer. British Journal of medicine and medical Research. DOI:10.9734/BJMMR/2014/12841.

13.        Naveenkumar C, Asokkumar S, Raghunandhakumar S, Jagan S, Anandakumar P, Augustine TA, Kamaraj S, Devaki T. Potent antitumor and antineoplastic efficacy of baicalein on benzo(a)pyrene-induced experimental pulmonary tumorigenesis. Fundam Clin Pharmacol. 2012 Apr;26(2):259-70. PubMed PMID: 21323996.

14.        Ramakrishnan G, Lo Muzio L, Elinos-Báez CM, Jagan S, Augustine TA, Kamaraj S, Anandakumar P, Devaki T. Silymarin inhibited proliferation and induced apoptosis in hepatic cancer cells. Cell Prolif. 2009 Apr;42(2):229-40. PubMed PMID: 19317806.

15.        Anandakumar P, Jagan S, Kamaraj S, Ramakrishnan G, Titto AA, Devaki T. Beneficial influence of capsaicin on lipid peroxidation, membrane-bound enzymes and glycoprotein profile during experimental lung carcinogenesis. J Pharm Pharmacol. 2008 Jun; 60(6):803-8. PubMed PMID:18498718.

16.        Ramakrishnan G, Elinos-Báez CM, Jagan S, Augustine TA, Kamaraj S, Anandakumar P, Devaki T. Silymarin downregulates COX-2 expression and attenuates hyperlipidemia during NDEA-induced rat hepatocellular carcinoma. Mol Cell Biochem. 2008 Jun;313(1-2):53-61. PubMed PMID: 18373278.

17.        Ramakrishnan G, Augustine TA, Jagan S, Vinodhkumar R, Devaki T. Effect of silymarin on N-nitrosodiethylamine induced hepatocarcinogenesis in rats. Exp Oncol. 2007 Mar;29(1):39-44. PubMed PMID: 17431387.

18.        Augustine TA, Sakthivel R, Ramakrishnan G, Devaki T. Squalene’s elicitation of cytoprotection against N-nitrosodiethylamine induced hepatocellular carcinoma is mediated through antioxidant mechanism. BMC Cancer (under communication).