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Dysregulated fatty acid metabolism is thought to be a hallmark of cancer, wherein fatty acids function both as an energy source and as signals for enzymatic and transcriptional networks contributing to malignancy. Fatty acid-binding protein 5 (FABP5) is an intracellular protein that facilitates transport of fatty acids and plays a role in regulating the expression of genes associated with cancer progression such as cell growth, survival, and metastasis. Overexpression of FABP5 has been reported to contribute to an aggressive phenotype and a poor survival correlation in several cancers. Therefore, inhibition of FABP5 is considered as a therapeutic approach for cancers. Phenotypic screening of a library of covalent compounds for selective sensitivity of cancer cells followed by medicinal chemistry optimization resulted in the identification of AUR104 with desirable properties. Chemoproteomic-based target deconvolution revealed FABP5 as the cellular target of AUR104. Covalent adduct formation with Cys43 of FABP5 by AUR104 was confirmed by mass spectrometry. Target occupancy studies using a biotin-tagged AUR104 demonstrated potent covalent binding to FABP5 in both cell-free and cellular conditions. Ligand displacement assay with a fluorescent fatty acid probe confirmed the competitive binding mode of AUR104 with fatty acids. Binding at the fatty acid site and covalent bond formation with Cys43 were also demonstrated by crystallography. Furthermore, AUR104 showed a high degree of selectivity against a broad safety pharmacology panel of enzymes and receptors. AUR104 exhibited potent anti-proliferative activity in a large panel of cell lines derived from both hematological and solid cancers with a high degree of selectivity over normal cells. Anti-proliferative activity in lymphoma cell lines correlated with inhibition of MALT1 pathway activity, cleavage of RelB/Bcl10 and secretion of cytokines, IL-10 and IL-6. AUR104 displayed desirable drug-like properties and dose-dependent oral exposure in pharmacokinetic studies. Oral dosing with AUR104 resulted in dose-dependent anti-tumor activity in DLBCL (OCI-LY10) and NSCLC (NCI-H1975) xenograft models. In a repeated dose MTD studies in rodents and non-rodents, AUR104 showed good tolerability with an exposure multiple of >500 over cellular EC50 for up to 8 hours. In summary, we have identified a novel covalent FABP5 inhibitor with optimized properties that showed anti-tumor activity in in vitro and in vivo models with acceptable safety profile. The data presented here strongly support clinical development of AUR104. Citation Format: Dinesh Chikkanna, Leena Khare Satyam, Sunil Kumar Pnaigrahi, Vinayak Khairnar, Manoj Pothuganti, Lakshmi Narayan Kaza, Narasimha Raju Kalidindi, Vijaya Shankar Nataraj, Aditya Kiran Gatta, Narasimha Rao Krishnamurthy, Sandeep Patil, DS Samiulla, Kiran Aithal, Vijay Kamal Ahuja, Nirbhay Kumar Tiwari, KB Charamannna, Pravin Pise, Thomas Anthony, Kavitha Nellore, Sanjeev Giri, Shekar Chelur, Susanta Samajdar, Murali Ramachandra. Discovery and preclinical evaluation of a novel covalent inhibitor of FABP5 for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1266.

Background: SMARCA2 (BRM) and SMARCA4 (BRG1) are two mutually exclusive DNA-dependent ATPases of the SWI/SNF complex, which function in mobilizing nucleosomes to regulate transcription, DNA replication/repair and chromosome dynamics. SMARCA4 is known to be mutated in number of cancers lacking targetable oncogenes, with SMARCA4-mutant patient population representing 10%-20% of NSCLC, 100% small cell ovarian cancer (hypercalcemic type), 28% skin cancer, 16% glioma and 14% colon cancer. Genetic studies have established the necessity of SMARCA2 for survival of tumor cells lacking SMARCA4. Although genetic silencing of SMARCA2 leads to potent anti-proliferative activity in SMARCA4-deficient cancer cell lines, pharmacological studies with a probe capable of binding to SMARCA2 and SMARCA4 bromodomains have failed to recapitulate such anti-proliferative effects. This prompted us to evaluate targeted protein degradation as an alternate approach to target SMARCA4 altered cancers. Methods and Results: A variety of hetero bi-functional molecules were synthesized by conjugating selective SMARCA2/4 bromodomain inhibitors with either VHL or CRBN E3-ligase specific ligands. Rational design approach guided by our proprietary ternary complex modeling algorithm, ALMOND (ALgorithm for MOdeling Neosubstrate Degraders) resulted in the identification of highly selective SMARCA2 degraders. The lead compound, AU-19820 showed > 10000-fold selectivity for SMARCA2 degradation versus other homologous proteins in tested cell lines. AU-19820 demonstrated potent anti-proliferative activity in SMARCA4 mutant but not in SMARCA2/4 proficient cell lines. This compound displayed favorable IV PK profile in rodents along with clean CYP profile. Additionally, the lead compound exhibited significant tumor growth inhibition in RERF-LC-A1 (SMARCA4 mutant lung cancer) xenograft model when dosed via i.v. route. Efficacious exposures were well tolerated with excellent tumor penetration. The compound also demonstrated moderate oral bioavailability in mouse. Efforts are in progress to improve this further by SAR modifications and exploiting prodrug approach. Conclusions: Potent and extremely selective SMARCA2 protein degraders were identified by conjugating SMARCA2/4 inhibitors with known VHL or CRBN ligands. SMARCA2 vs SMARCA4 selectivity handles have been very well explored with expandable SAR. Lead compound also displayed a synthetic lethality phenotype in SMARCA4 mutant cancer models while sparing SMARCA2/4 proficient ones. Further optimization of the oral bioavailability and evaluation of efficacy through oral route as well as intermittent IV dosing are planned. Citation Format: Chandrasekhar Abbineni, Leena Khare Satyam, Bilash Kuila, Ashok Ettam, Khaji Abdul Rawoof, Sreevidya MR, Sandeep Vitthal Dukare, Suraj T. Gore, Rakesh P. Nankar, Vijay Kamal Ahuja, Charamanna KB, Megha Goyal, Kiran Aithal, Samiulla DS, Subhendu Mukherjee, Thomas Antony, Sanjeev Giri, Shekar Chelur, Kavitha Nellore, Girish Daginakatte, Murali Ramachandra, Susanta Samajdar. Orally bioavailable SMARCA2 degraders with exceptional selectivity and potency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1144.

Gallbladder cancer is a lethal malignancy of hepato-biliary system with high incidence in North India, especially along gangetic plain. The let-7 microRNAs play a key role in regulating KRAS expression and a polymorphism in 3′ untranslated region (rs61764370, T/G) of KRAS leads to its higher expression. This polymorphism is known to be associated with increased risk and prognosis of various cancers but its association with gallbladder cancer has not been evaluated. To address this research question, we evaluated whether rs61764370 variant is associated with gallbladder cancer susceptibility and clinical outcomes. In present case–control study, we enrolled 541 patients with gallbladder malignancy and 307 controls. Genomic DNA was obtained from peripheral blood and genotyping was performed using Taqman allelic discrimination assay. Heterozygous (TG) individuals are at a significant higher risk for GBC as compared with wild genotype (TT) (p = 0.007, odds ratio = 2.56, 95 % CI 1.27–5.18). At allelic level, allele G has significant higher risk for GBC as compared with T allele (p = 0.008, odds ratio = 2.5, 95 % CI 1.25–5.01). Survival analysis reveals decrease in overall survival for heterozygous genotype (p

Pioneering success of antibodies targeting immune checkpoints such as PD-1 and CTLA4 has opened novel avenues for cancer immunotherapy. Along with impressive clinical activity, severe immune-related adverse events (irAE) due to the breaking of immune self-tolerance are becoming increasingly evident in antibody-based approaches. As a strategy to better manage severe adverse effects, we set out to discover an antagonist targeting PD-1 signaling pathway with a shorter pharmacokinetic profile. Herein, we describe a peptide antagonist NP-12 that displays equipotent antagonism toward PD-L1 and PD-L2 in rescue of lymphocyte proliferation and effector functions. In preclinical models of melanoma, colon cancer, and kidney cancers, NP-12 showed significant efficacy comparable with commercially available PD-1–targeting antibodies in inhibiting primary tumor growth and metastasis. Interestingly, antitumor activity of NP-12 in a preestablished CT26 model correlated well with pharmacodynamic effects as indicated by intratumoral recruitment of CD4 and CD8 T cells, and a reduction in PD-1+ T cells (both CD4 and CD8) in tumor and blood. In addition, NP-12 also showed additive antitumor activity in preestablished tumor models when combined with tumor vaccination or a chemotherapeutic agent such as cyclophosphamide known to induce “immunologic cell death.” In summary, NP-12 is the first rationally designed peptide therapeutic targeting PD-1 signaling pathways exhibiting immune activation, excellent antitumor activity, and potential for better management of irAEs.

Matriptase is a cell-surface trypsin-like serine protease of epithelial origin, which cleaves and activates proteins including hepatocyte growth factor/scatter factor and proteases such as uPA, which are involved in the progression of various cancers. Here we report a fragment-linking approach, which led to the discovery of O-(3-carbamimidoylphenyl)-l-serine amides as potent matriptase inhibitors. The co-crystal structure of one of the potent inhibitors, 6 in complex with matriptase catalytic domain validated the working hypothesis guiding the development of this congeneric series and revealed the structural basis for matriptase inhibition. Replacement of a naphthyl group in 6 with 2,4,6-tri-isopropyl phenyl resulted in 10 with improved matriptase inhibition, which exhibited significant primary tumor growth inhibition in a mouse model of prostate cancer. Compounds such as 10, identified using a fragment-linking approach, can be explored further to understand the role of matriptase as a drug target in cancer and inflammation.

SMARCA2/BRM and SMARCA4/BRG1 are the mutually exclusive DNA-dependent ATPases within the SWI/SNF complexes, which function in mobilizing nucleosomes to regulate transcription, DNA replication and repair, and higher-order chromosome dynamics. SMARCA4 is mutated in a number of cancers, which generally lack targetable oncogenes. Genetic silencing studies have established a requirement of SMARCA2 for survival of tumor cells lacking SMARCA4. SMARCA4-deficient patient population represents 10%-20% of NSCLC cases, ∼5% pancreatic cancer patients and ∼10% ovarian cancer patients where SMARCA2 is overexpressed. Interestingly, SMARCA4 is highly expressed without mutation in certain tumor types, where overexpression contributes to increased proliferation and survival. SMARCA4 knockdown in these tumors leads to inhibition of proliferation and also increase sensitivity to known chemotherapeutic agents, supporting the validity of targeting SMARCA4. Although genetic silencing of SMARCA2 leads to potent anti-proliferative activity in SMARCA4-deficient cancer cell lines, pharmacological studies with a probe capable of binding to SMARCA2 and SMARCA4 bromodomain have failed to show such an anti-proliferative phenotype. These findings triggered us to evaluate chemical degradation as an alternate approach to target SMARCA2/4 altered cancers. Optimization of bifunctional molecules with binding moieties for SMARCA2/4 and E3 ligase to induce proteasome-mediated degradation resulted in the identification of selective SMARCA2 and SMARCA4 degraders. These degraders showed selectivity against other bromodomain containing proteins such as BRD4, CBP and p300 in Western blot analysis. Functional analysis of a preferential SMARCA2 degrader in a panel of cell lines indicated a potent anti-proliferative activity in the context of SMARCA4 mutation. Additionally, these compounds displayed acceptable drug-like properties including solubility, metabolic stability and pharmacokinetics in mice. Dose-dependent tumor growth inhibition was observed in a SMARCA4-deficient lung cancer xenograft model and a syngeneic model of lymphoma at well-tolerated doses. Observed efficacy was correlated with the target degradation in the tumor supporting the potential to further develop them for cancer therapy. Based on the reported vulnerability of SMARCA4-deficient cell lines of diverse tumor origin to agents targeting PARP, PI3K/AKT and EZH2, combination effects with SMARC2 degrader are being interrogated. Citation Format: Leena Khare Satyam, Sanjita Sasmal, Manoj K. Pothuganti, Sreevidya M.R., Ashokk Ettam, Sireesha Nunna, Marla Roshaiah, Shankaraiah Chithaluru, Rangarao Pallepati, Amitkumar A. Pawar, Leela P. Narukulla, Raghunadh A. Sripathi, Suraj Tgore, Rakesh P. Nankar, Charamanna KB, Nagesh Gowda S, Kiran Aithal, Samiulla DS, Subhendu Mukherjee, Shekar Chelur, Kavitha Nellore, Girish Daginakatte, Murali Ramachandra, Susanta Samajdar. Anti-tumor efficacy of SMARCA degraders in pre-clinical models of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3844.

e15655 Background: Gallbladder cancer is the most common malignancy of the biliary tract and endemic in Northern India. The prognosis of gallbladder cancer remains dismal despite any treatment because of late presentation and aggressive tumor biology. Cholecystokinin (CCK) is present in abundance in gallbladder tissue and mediates function through two structurally related receptors, the type A (CCKAR) and B (CCKBR) receptors. Previously, heterodimerization of CCKAR and CCKBR receptor has been demonstrated in vitro, and was shown to bind natural agonists normally but exhibited agonist stimulated cellular signaling, promoting cell growth. In this study we examined the expression level of CCKAR and CCKBR in resected gallbladder tissue samples and also attempted to determine the dimerization status of CCKAR and CCKBR receptors in gallbladder cancer comparing them to cholelithiasis and normal gallbladder tissue. Methods: Tissue samples from resected normal gallbladder (n = 10), cholelithiasis (n = 25) and gallbladder cancer (n = 25)) were evaluated for the expression of CCKAR and CCKBR by immunohistochemistry. Determination of CCKAR and CCKBR expression was also done by western blot in representative samples from each of these tissue types while their dimerization status was evaluated by immunoprecipitation. Results: By Immunohistochemistry technique, positive CCKAR expression was observed in 80% gallbladder cancer, 84% cholelithiasis, 90% normal gallbladder, P = 0.76. However significantly higher expression of CCKBR was noted in gallbladder cancer (60%) as compared to cholelithiasis (25%) and normal gallbladder (20%), P = 0.013. We also observed progressively increasing level of expression of CCKAR and CCKBR in gallbladder cancer as compared to cholelithiasis and normal gallbladder by western blot. In addition, we also observed a higher level of heterodimer formation in gallbladder cancer compared to cholelithiasis and normal gallbladder using immunoprecipitation technique. Conclusions: Our results provide the first evidence of increasing trend of heterodimerization of CCKAR and CCKBR in gallbladder cancer which has potential clinical and therapeutic significance.

The SWI/SNF complexes consist of one of two mutually exclusive DNA-dependent ATPases, BRG1/SMARCA4 or BRM/SMARCA2, together with core and accessory subunits that function in mobilizing nucleosomes to regulate transcription, DNA replication and repair, and higher-order chromosome dynamics. SMARCA2 (BRM) and SMARCA4 (BRG1), contain a bromodomain and an ATPase domain. The mammalian SWI/SNF complex functions as a tumor suppressor in many human malignancies. In addition to an essential role in pluripotency and development, genetic lesions of SWI/SNF complexes have been strongly linked to cancer development as components are mutated in many cancers. In some tumor types, mutations within the SWI/SNF complex lead to context specific vulnerabilities such as the requirement of SMARCA2 for survival of tumour cells lacking SMARCA4. This finding of SMARCA2/4 synthetic lethal relationship translates in vivo which emphasizes SMARCA2 as a promising therapeutic target for the treatment SMARCA4-mutant cancers. Moreover, the SMARCA4-deficient patient population generally lacks targetable oncogenes (such as mutant EGFR or ALK translocations), which further emphasizes the potential of developing SMARCA2 inhibitors. Previously, contrary to genetic silencing of SMARCA2 leading to potent anti-proliferative activity in SMARCA4-deficient cancer cell lines, pharmacological studies with a cell permeable probe capable of binding to SMARCA2 and SMARCA4 have failed to show such an anti-proliferative phenotype. These findings support that the ATPase domain, but not the bromodomain of SMARCA2, is the tractable therapeutic target for SMARCA4-deficient cancer. Significant hurdle in developing potent and selective ATPase inhibitors that must compete with intracellular concentrations of ATP (2-10 mM) triggered us to evaluate chemical degrader as an alternate approach. Herein we report the identification and characterization of potent SMARCA2/4 degraders. Design and SAR-based optimization of bifunctional molecules with binding moities for SMARCA2/4 and E3 ligase to induce proteosome-mediated degradation yielded compounds that potently degraded SMARCA2 alone or both SMARCA2 and SMARCA4. Selective binding to SMARCA2/4 was confirmed in biochemical assays followed by assessement of their cellular degradation potency in Western blot analysis. Selective degradation of SMARCA2/4 over other bromodomain containing protein such as BRD4 and CBP/p300 was also observed. Functional analysis of these compounds in a panel of cell lines indicated that the degradation of SMARCA2 or SMARCA2/4 resulted in potent anti-proliferative activity in selected cell lines that was not strictly dependent upon SMARCA4 status. Additionally, these compounds displayed reasonable drug-like properties including solubility, metabolic stability and pharmacokinetics in mice supporting their potential to fully evaluate the impact of SMARCA2/4 degradation and to develop them as a novel therapeutic approach. Citation Format: Sanjita Sasmal, Leena K. Satyam, Manoj K. Pothuganti, Ashokk Ettam, Sireesha Nunna, Mohammed A. Shareef, Sreevalsam Gopinath, Subhendu Mukherjee, Murali Ramachandra, Susanta Samajdar. Identification of SMARCA2/4 degraders for the treatment of SMARCA4-mutant and other cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-258.

Background: Cyclin-dependent kinase 7 (CDK7) is an important constituent of the cellular transcriptional machinery, where it phosphorylates the C-terminal domain (CTD) of RNAP polymerase II (RNAPII). Because many tumor types are critically dependent on transcription for maintenance of their oncogenic state, pharmacologic modulation of CDK7 kinase activity is considered as an approach to treat cancer. Experimental Procedures: Multiple series of covalent CDK7 inhibitors were identified by iterative medicinal chemistry efforts and SAR-based approach. These compounds were optimized towards attaining good physicochemical properties, high potency, good selectivity, and desirable pharmacokinetic profile to achieve antitumor activity. Summary: We have now identified a highly selective and orally bioavailable covalent CDK7 inhibitor AU-002 as a preclinical candidate. AU-002 is highly potent in inhibiting CDK7 in biochemical assays with robust engagement of CDK7 in cellular context. In a broad panel of 372 kinases, AU-002 shows excellent selectivity for CDK7. AU-002 exhibits excellent drug-like characteristics including solubility, permeability, metabolic stability, and good oral bioavailability. Oral treatment of this compound in a xenograft model resulted in dose-dependent tumor growth inhibition in AML xenograft model and with complete tumor regression at well-tolerated doses. Potent inhibition of tumor growth was accompanied by complete target engagement and suppression of pS5RNAPII in a parallel PK-PD study. Efficacy studies in additional xenograft models, advanced DMPK, and toxicity studies are ongoing for this compound. Conclusion: We have identified a novel and highly selective CDK7 covalent inhibitor candidate with good oral bioavailability that shows excellent efficacy in an AML xenograft model. Findings presented here support further development of AU-002 for the treatment of cancer. Citation Format: Ramulu Poddutoori, Leena Khare Satyam, Subhendu Mukherjee, Sivapriya Marappan, Sreevalsam Gopinath, Charamanna KB, Lakshmi Narayana Kaza, Manoj Kumar Pothuganti, Sujatha Rajagopalan, Sasirekha Sivakumar, Bharath E N, Aravind A B, Amith A, Ravindra M V, Suraj Tgore, Thomas Antony, Chetan Pandit, Shekar Chelur, Girish Daginakatte, Murali Ramachandra, Susanta Samajdar. Identification of a novel highly selective and orally bioavailable preclinical candidate for CDK7 covalent inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B164.

Cyclin-dependent kinase 7 (CDK7) is an important constituent of the cellular transcriptional machinery, where it phosphorylates the C-terminal domain (CTD) of RNAP polymerase II (RNAPII). Because many tumor types are critically dependent on transcription for maintenance of their oncogenic state, pharmacological modulation of CDK7 kinase activity is considered as an approach to treat cancer. Multiple series of covalent CDK7 inhibitors were identified by iterative medicinal chemistry efforts and SAR based approach. These compounds were optimized towards attaining good physicochemical properties, high potency, good selectivity and desirable pharmacokinetic profile to achieve anti-tumor activity. We have now identified a pre-clinical candidate AU-BGB-002 which is highly potent in inhibiting CDK7 in biochemical as well as cellular assays while fully efficiently engaging the target. In a panel of kinases, AU-BGB-002 shows selectivity for CDK7. A panel of cell lines derived from a diverse set of indications are sensitive to AU-BGB-002. AU-BGB-002 exhibits excellent drug-like characteristics including solubility, permeability, metabolic stability and good oral bioavailability. When tested in a xenograft model, AU-BGB-002 treatment resulted in dose dependent tumor growth inhibition in AML xenograft model with tumor stasis at a dose of 10 mg/kg. Potent inhibiton of tumor growth was accompanied by complete target engagement and suppression of pS5RNAPII RNAPolII Ser5 phosphorylation in a parallel PK-PD study. Efficacy studies in additional xenograft models, advanced DMPK and toxicity studies are ongoing for this compound. In summary, we have identified a novel and selective CDK7 covalent inhibitor candidate with desirable drug-like properties that shows excellent efficacy in an AML xenograft model. Findings presented here support further development of AU-BGB-002 for the treatment of cancer. Citation Format: Leena K. Satyam, Ramulu Poddutoori, Subhendu Mukherjee, Sivapriya Marappan, Sreevalsam Gopinath, Aravind Basavaraju, Lakshmi Narayana Kaza, Manoj Kumar Pothuganti, Shilpa Nayak, Nandish C, Amith A, Ravindra MV, Dabbeeru Madhu Babu, Nagaraju A, Suraj Tgore, Thomas Antony, Chetan Pandit, Murali Ramachandra, Shekar Chelur, Girish Daginakatte, Susanta Samajdar. Identification of a novel preclinical candidate for CDK7 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-317. doi:10.1158/1538-7445.AM2017-LB-317

Gallbladder Cancer (GBC) is the most common malignancy of the biliary tract with very high incidence in Northern India, especially around Gangetic belt. Late diagnosis and poor prognosis are the important features of this disease. For improving treatment strategies and outcomes, novel non invasive prognostic biomarkers are needed. Variations in peripheral blood mitochondrial DNA (mtDNA) copy number in various cancers are widely studied but information in relation to gallbladder malignancy is unknown. In this pilot study, we investigated leucocytes mtDNA copy number variation in relation to risk and prognosis of GBC. A total of 300 histologically confirmed or fine needle aspiration cytology proven GBC patients were recruited along with equal number of age and sex matched controls. The mtDNA copy number was measured by real-time polymerase chain reaction through absolute quantification using SYBR green chemistry. We also explore prognostic utility of mtDNA content in relation to overall survival of GBC patients. Statistical analysis was done by Student’s test and survival data was compared using Kaplan Meier method. Two tailed p value less than 0.05 was considered to be significant. We observed high mtDNA content in cases as compared with controls (mean±standard deviation, 1.21±0.88 vs. 1.02±0.76). Higher mtDNA copy number was significantly associated with increased gallbladder cancer risk (p=0.005). In this study, we compared overall survival (OS) for gallbladder cancer patients in relation to mtDNA copy number median value (median, 1.12). Kaplan Meir survival curve predicts decrease in OS (p=0.03, Hazard ratio=2.54; 95% CI = 1.79-5.86) for GBC patients in relation to high mtDNA content (>median) as compared with low mtDNA copy number ( This study supports an association between increased mtDNA copy number with susceptibility to gallbladder cancer in this endemic belt. Future studies are warranted to confirm these findings and to elucidate the biological role of mtDNA copy number in relation to gallbladder cancer. Citation Format: Abhijit Chandra, Hasan R. Kazmi, Leena K. Satyam, Saket Kumar. Association of mitochondrial DNA copy number variation in peripheral blood leucocytes with risk and prognosis of gallbladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4729. doi:10.1158/1538-7445.AM2017-4729

Diffuse large B cell lymphoma (DLBCL), which accounts for 25% of all lymphomas cases, has been classified into molecular subtypes including germinal center B cell like (GCB) DLBCL, activated B cell-like (ABC) DLBCL, and primary mediastinal B cell lymphoma (PMBL). Among these subtypes, patients with ABC-DLBCLs have the worst prognosis because of the high chemo-resistance, and require effective therapies. Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 (MALT1) protease activity is linked to the pathogenesis of ABC-DLBCL. Therefore, a focused library of covalent compounds selected based on molecular docking on the reported crystal structure was screened for selective sensitivity to ABC-DLBCL, but not GCB-DLBCL cell lines. Optimization of initial hits resulted in the identification of lead compounds with an anti-proliferative EC50 of 10 over cellular EC50 for up to 8 hours. The lead compounds showed dose-dependent tumor growth inhibition in a xenograft model upon oral dosing. In summary, we have identified novel and potent MALT1 inhibitors capable of selectively inhibiting proliferation of DLBCL cell lines with optimized drug-like properties including oral bioavailability. The data presented here strongly support further development of these compounds for DLBCL and other indications. Citation Format: Leena Khare Satyam, Dinesh Chikkanna, Vinayak Khairnar, Manoj Pothuganti, Sunil Panigrahi, Anirudha Lakshminarasimhan, Narasimha Rao, Wesley Balasubramanian, Sandeep Patil, Sreevalsam Gopinath, Gunta Upendra, Jwala Nagaraj, Kiran Aithal, Vijay Ahuja, Sanjeev Giri, Chetan Pandit, Murali Ramachandra. Potent small molecule compounds that selectively inhibit proliferation of ABC-DLBCL cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5108. doi:10.1158/1538-7445.AM2017-5108

Matriptase is a serine protease implicated in cancer invasion and metastasis. Expression of matriptase is frequently dysregulated in human cancers and matriptase has been reported to activate latent growth factors such as hepatocyte growth factor/scatter factor, and proteases such as urokinase plasminogen activator suggesting that matriptase inhibitors could have therapeutic potential in treatment of cancer. Here we report a structure-based approach which led to the discovery of selective and potent matriptase inhibitors with benzene as central core having 1,3,5 tri-substitution pattern. X-ray crystallography of one of the potent analogs in complex with matriptase revealed strong hydrogen bonding and salt-bridge interactions in the S1 pocket, as well as strong CH-π contacts between the P2/P4 cyclohexyl and Trp215 side-chain. An additional interaction of the pendant amine at cyclohexyl with Gln175 side-chain results in substantial improvement in matriptase inhibition and selectivity against other related serine proteases. Compounds 15 and 26 showed tumor growth inhibition in a subcutaneous DU-145 prostate cancer mouse model. These compounds could be useful as tools to further explore the biology of matriptase as a drug target.

Matriptase belongs to trypsin-like serine proteases involved in matrix remodeling/degradation, growth regulation, survival, motility, and cell morphogenesis. Herein, we report a structure-based approach, which led to the discovery of sulfonamide and amide derivatives of pyridyl bis(oxy)benzamidine as potent and selective matriptase inhibitors. Co-crystal structures of selected compounds in complex with matriptase supported compound designing. Additionally, WaterMap analyses indicated the possibility of occupying a distinct pocket within the catalytic domain, exploration of which resulted in >100-fold improvement in potency. Co-crystal structure of 10 with matriptase revealed critical interactions leading to potent target inhibition and selectivity against other serine proteases.

KRAS is the frequently mutated isoform in RAS driven cancers. The G12C mutation is more predominantly associated with various tumor types over other changes in K-Ras. Although direct targeting of RAS is very challenging, it is possible to selectively target G12C mutant K-Ras using a covalent approach. Mutant specific covalent inhibitors with high selectivity against wild type K-Ras and other GTPases are expected to lead to efficacy with a very high degree of tolerability. Here, we report identification of lead compounds from two distinct chemical series that selectively target K-Ras G12C. Molecular modeling based on the reported crystal structures aided in the identification of these compounds. Covalent binding of the lead compounds to K-Ras G12C was demonstrated by MALDI-TOF. Lead compounds were potent in selectively inhibiting proliferation of cell lines with K-Ras G12C mutation but not with wild type K-Ras. The anti-proliferative activity of the lead compounds correlated well with their potency in a cellular mechanistic assay. Lead compounds from both series exhibited excellent drug-like properties including solubility, metabolic stability, permeability lack of CYP inhibition and desired exposure in pharmacokinetic studies. In a xenograft model of NSCLC, the lead compound demonstrated dose-dependent tumor growth inhibition with excellent tolerability upon oral dosing. In summary, we have identified a novel, potent and selective K-Ras G12C inhibitor with optimized drug-like properties including oral bioavailability and efficacy in a NSCLC derived xenograft model. Toxicity evaluation is ongoing towards progressing the lead compound to the clinic. Citation Format: Leena Khare Satyam, Dinesh Chikkanna, Aswani K. G, Vinayak V. Khairnar, Sreekanth Reddy, Vakkapatla Durgaprasad, Kowju Radhakrishna, Sunil K. Panigrahi, Anuradha Ramanathan, Kumari Mahasweta, Anirudha Lakshminarasimhan, Narasimha R. K, Vinutha R, Sreevalsam Gopinath, Suryakant Kumar, Mubarak H. Shah, Raghuveer Ramachandra, Kiran A. B, Chetan Pandit, Murali Ramachandra. Identification of novel covalent inhibitors of K-Ras G12C that are efficacious in a xenograft model of NSCLC. [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 339.

Cyclin-dependent kinase 7 (CDK7) is an important constituent of the cellular transcriptional machinery, where it phosphorylates the C-terminal domain (CTD) of RNAP polymerase II (RNAPII). Because many tumor types are critically dependent on transcription for maintenance of their oncogenic state, pharmacological modulation of CDK7 kinase activity is considered as an approach to treat cancer. Multiple series of CDK7 inhibitors were identified by iterative medicinal chemistry efforts and SAR based approach. Early compounds were optimized towards attaining good physicochemical properties, high potency, good selectivity and desirable pharmacokinetic profile to achieve anti-tumor activity. We have identified compounds from two distinct chemical series that are highly potent in inhibiting CDK7 in biochemical assays. These inhibitors demonstrate time-dependent inhibition of CDK7 indicating covalent nature of binding. The compounds showed potent anti-proliferative activity in cell lines derived from various tumor types and this was accompanied by CDK7 modulation in cells as monitored by pS5RNAPII levels. They have excellent drug-like characteristics including solubility, permeability, metabolic stability and good oral bioavailability. In a broad panel of kinases (332 kinase), selected compounds from both series showed good selectivity profile. Tolerability and efficacy studies are ongoing with selected early leads to test their impact on tumor growth inhibition in xenograft models. We have identified novel and selective CDK7 covalent inhibitors from two series with desirable drug-like properties, which are being evauated for anti-tumor activity in xenograft models. Citation Format: Ramulu Poddutoori, Leena K. Satyam, Girish Daginakatte, Subhendu Mukherjee, Sivapriya Marappan, Sreevalsam Gopinath, Raghuveer Ramachandra, Anirudha Lakshminarasimhan, Manoj Pothuganti, Shilpa Nayak, Nandish C, Chandranath Naik, Ravindra MV, Madhu Dabbeeru, Thomas Antony, Chetan Pandit, Murali Ramachandra, Shekar Chelur, Susanta Samajdar. Potent and selective inhibition of CDK7 by novel covalent inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C190.

Antibodies to immune checkpoint receptors have revolutionized the outlook of cancer therapy by achieving highly durable clinical responses. However, along with impressive clinical activity (response rate of ∼25% with either anti-CTLA4 or anti-PD1 as single agent, but > 50% with a combination), severe immune-related adverse events (irAEs) due to the breaking of immune self- tolerance (25-30% with anti-CTLA4 and up to 15-17% with anti-PD1) are becoming increasingly evident. Sustained target inhibition as a result of a long half-life (>15-20 days) and >70% target occupancy for months are likely contributing to severe irAEs observed in the clinic with antibodies targeting immune checkpoint proteins. The antibodies targeting PD-1 signaling pathway are either based on receptor (PD-1) or its ligands (PD-L1 & PDL2). It has also been reported that PDL1 also interacts with B7-1(CD80) resulting in negative T-cell regulation. Our efforts are therefore focused on (a) developing a first in class peptide therapeutic inhibiting all three reported interaction of PD-1 pathway to improve the anti-tumor immune response (b) developing immune checkpoint blockers with potent anti-tumor activity but with a shorter pharmacokinetic profile as a strategy to better manage severe irAEs. Peptide antagonist AUR-012, constructed with elements from human PD-1 receptor, displayed equipotent antagonism towards PD-L1 and PD-L2 with potent activity in rescue of lymphocyte proliferation and effector functions. Additionally crosslinking study to evaluate the effect of AUR-12 on B7.1 - PDL-1 interaction supports the finding that AUR-012 prevents the interaction of B7.1 with PD-L1. Rescue of proliferation of immune cells analyzed upon stimulation with anti-CD3/anti-CD-28 indicated a complete rescue of CD4+ and CD8+ T cells and a complete suppression of regulatory T cells. Sustained activation of circulatory immune cells and their ability to secrete IFN-γ up to 72 h indicate that pharmacodynamic effects persist even after the clearance of AUR-012 in animal models, thus supporting a dosing interval of up to 3 days. In models of melanoma, breast, kidney and colon cancers, AUR-012 showed efficacy in inhibition of both primary tumor growth and metastasis. Additionally, anti-tumor activity of AUR-012 in a pre-established CT26 model correlated well with pharmacodynamic effects as indicated by intratumoral recruitment of CD4+ and CD8+ T cells, and a reduction in PD1+ T cells (both CD4+ & CD8+) in tumor and blood. In 14-day repeated dose toxicity studies, AUR -012 was well tolerated at 100x of the efficacious doses. These findings demonstrating inhibition of all PD-1/ PD-1 ligands mediated co-inhibitory signaling pathway and the observed correlation between anti-tumor activities with the modulation of specific T-cell populations support further development of AUR-012 in the clinic. Citation Format: Murali Ramachandra, Pottayil G. Sasikumar, Rajeev K. Shrimali, Sreenivas Adurthi, Raghuvir Ramachandra, Leena K. Satyam, Amit A. Dhudashia, Samiulla Dhodheri, K B. Sunilkumar. Peptide therapeutic inhibiting PD1 immune checkpoint ligands: Potential for greater anti-tumor immune response and better management of immune-related adverse events. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5022. doi:10.1158/1538-7445.AM2014-5022

Recent breakthroughs in achieving highly durable clinical responses via immunotherapy to inhibit immune checkpoint proteins including CTLA4 and PD1 have revolutionized the outlook for cancer therapy. However, along with impressive clinical activity (response rate of ∼25% with either anti-CTLA4 or anti-PD1 as single agent, but more than 50% with a combination), immune-related toxicities due to the breaking of immune self-tolerance (25-30% with anti-CTLA4 and up to 15-17% with anti-PD1) are becoming increasingly visible. Sustained inhibition of the signaling as a result of a very long half-life (>15-20 days) and >70% target occupancy for months are likely contributing to severe adverse effects observed in clinical trials with antibodies targeting immune checkpoint proteins. Our efforts are therefore focused towards developing novel immune checkpoint blockers with potent antitumor activity but with a shorter pharmacokinetic profile as a strategy to better manage severe adverse effects. Herein we report our findings from the pharmacodynamic characterization of a novel peptide inhibitor of the PD1 signaling that displays desired pharmacokinetic and efficacy profile. Peptide antagonist AUR-012, constructed with elements from human PD-1 ectodomain, displayed equipotent antagonism towards PD-L1 and PD-L2 with potent activity in rescue of lymphocyte proliferation and effector functions. Rescue of proliferation of immune cell subset populations analyzed in vitro using anti-CD3/anti CD-28 stimulation indicated that the functional capacity of T-cells has been reinvigorated by AUR-012 resulting in a complete rescue of CD4+ and CD8+ T cell proliferation in the presence of PD-L1. Similar effect in the rescue of proliferation was also observed for B-cells and NK cells. Interestingly, the proliferation of CD4+, FOxP3+ T cells was completely abolished with AUR-012 treatment indicating a complete suppression of regulatory T cells. Sustained activation of circulatory immune cells and their ability to secrete IFN-γ up to 72 h indicate that pharmacodynamic effects persist even after the clearance of AUR-012 in animal models, thus supporting a dosing interval of up to 3 days irrespective of the short half-life of the peptide. In preclinical models of melanoma, breast, kidney and colon cancers, AUR-012 showed superior showed superior efficacy compared to therapeutic agents currently used in the clinic in inhibition of both primary tumor growth and metastasis. Additionally, antitumor activity of AUR-012 in a pre-established CT26 model correlated well with pharmacodynamic effects as indicated by intratumoral recruitment of CD4+ and CD8+ T cells, and a reduction in PD1+ T cells (both CD4+ & CD8+) in tumor and blood. In conclusion, the observed correlation between antitumor activities with the modulation of specific T-cell populations supports the use of immunophenotyping as a potential therapeutic biomarker strategy in the planned clinical studies with AUR-012. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B294. Citation Format: Murali Ramachandra, Pottayil G. Sasikumar, Rajeev K. Shrimali, Sreenivas Adurthi, Raghuveer Ramachandra, Leena K. Satyam, Amit A. Dhudashiya, Dodheri S. Samiulla, K B Sunilkumar. Antitumor efficacy of AUR-012, a peptide antagonist of the PD1 immune checkpoint pathway, correlates well with the modulation of specific T-cell populations. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B294.

Pioneering success of anti-CTLA4 antibody (Ipilimumab) and the impressive clinical data on agents that target PD-1 or its ligands have opened novel avenues in the area for caner immunotherapy. However, along with impressive clinical activity, immune-related toxicities have also been observed in significant (25-30% with anti-CTLA4 and up to 15% with anti-PD1) patient population. Sustained inhibition of PD-1 signaling as a result of a very long half-life (>15-20 days) and >70% target occupation for months are likely contributing to severe adverse effects observed in clinical trials with antibodies targeting PD-1 signaling. In the present study, we sought to characterize in detail a novel peptide antagonist of the PD1 signaling providing desired anti-tumor efficacy with shorter exposure and thus transient immune activation for effective management of severe adverse effects. Design of peptide to disrupt PD-1 signalling pathway was carried out by selecting individual fragments from PD-1 ectodomain reported to participate in binding. Sequences critical for ligand-receptor interaction were identified and combined in a non-linear fashion. The strategy resulted in a novel peptide, AUR-012 (29-mer), which displayed equipotent antagonism in disruption of PD1-PDL1/2 interaction and highly effective in restoration of proliferation and effector functions of mouse splenocytes and monkey and human PBMCs using species specific ligands. In preclinical models of melanoma, breast, kidney and colon cancers, AUR-012 showed superior showed superior efficacy compared to therapeutic agents currently used in the clinic in inhibition of both primary tumour growth and metastasis. Additionally, AUR-012 showed additive anti-tumor activity in a pre-established tumor models when combined with tumor vaccination or a chemotherapeutic agent such as cyclophosphamide known to induce “immunological cell death”. Anti-tumoral activity was correlated with increased drug distribution within the tumor while promoting intratumoral recruitment of CD4+ and CD8+ T cells. Furthermore, AUR -012 was well tolerated in 14 days repeated dose toxicity studies at 100x efficacious doses. The above findings demonstrating equipotent activity antagonism of both PD-L1 and PD-L2 signaling coupled with impressive efficacy in preclinical models support further development of AUR-012 for clinical use. Citation Format: Pottayil G. Sasikumar, Leena K. Satyam, Rajeev Shrimali, Raghuveer Ramachandra, Ketha A. Reddy, Adurthi Sreenivas, Amit Dhudashia, Dodderi S. Samiulla, Murali Ramachandra. Equipotent antagonism, transient immune activation and excellent antitumor efficacy with a peptide inhibitor of PD-1 immune check point pathway. [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 1231. doi:10.1158/1538-7445.AM2013-1231

Meeting abstracts Recent advances in achieving highly durable clinical responses via inhibition of immune checkpoint proteins including CTLA-4 and PD1 have revolutionized the outlook for cancer therapy. However, along with impressive clinical activity (response rate of ~25% with either anti-CTLA-4

Programmed cell death-1 (PD-1), an immunoreceptor belonging to the CD28 family, plays an important role in negatively regulating immune responses. Blocking of PD-1 signalling pathway has been shown to result in restoration of defective immune cell functions in cancer and chronic infections. PD-1 targeted therapies in the ongoing clinical trials are based on either antibodies or fusion proteins. To exploit unique advantages of peptides over antibodies or fusion proteins towards addressing the limitations of the current clinical candidates, herein we report a peptide based strategy to block the PD-1 signaling pathway. Sequences critical for ligand-receptor interaction were identified and combined in a non-linear fashion. The strategy resulted in a novel peptide, AUR-012 (29-mer), which displayed sub-nanomolar potency in disruption of PD1-PDL1/2 interaction, and highly effective restoration of proliferation and effector functions of splenocytes and PBMCs. In vivo studies demonstrated an excellent PK-PD correlation with sustained PD for >24 h. In preclinical models of melanoma, breast and kidney cancers, AUR-012 showed superior efficacy compared to therapeutic agents currently used in the clinic in inhibition of both primary tumor growth and metastasis. Interestingly, dosing once in three days was equally efficacious as once a day dosing with no signs of overt toxicity and generation of neutralizing activity. These findings support further development of AUR-012 for potential clinical use. 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 2850. doi:1538-7445.AM2012-2850