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Phosphorylation is an important regulatory mechanism of protein activity in cells. Studies in various cancers have reported perturbations in kinases resulting in aberrant phosphorylation of oncoproteins and tumor suppressor proteins. In this study, we carried out quantitative phosphoproteomic analysis of gastric cancer tissues and corresponding xenograft samples. Using these data, we employed bioinformatics analysis to identify aberrant signaling pathways. We further performed molecular inhibition and silencing of the upstream regulatory kinase in gastric cancer cell lines and validated its effect on cellular phenotype. Through an ex vivo technology utilizing patient tumor and blood sample, we sought to understand the therapeutic potential of the kinase by recreating the tumor microenvironment. Using mass spectrometry-based high-throughput analysis, we identified 1,344 phosphosites and 848 phosphoproteins, including differential phosphorylation of 177 proteins (fold change cut-off ≥ 1.5). Our data showed that a subset of differentially phosphorylated proteins belonged to splicing machinery. Pathway analysis highlighted Cdc2-like kinase (CLK1) as upstream kinase. Inhibition of CLK1 using TG003 and CLK1 siRNA resulted in a decreased cell viability, proliferation, invasion and migration as well as modulation in the phosphorylation of SRSF2. Ex vivo experiments which utilizes patient’s own tumor and blood to recreate the tumor microenvironment validated the use of CLK1 as a potential target for gastric cancer treatment. Our data indicates that CLK1 plays a crucial role in the regulation of splicing process in gastric cancer and that CLK1 can act as a novel therapeutic target in gastric cancer.

The prognosis of AML is generally poor, with 5-year survival rate of 25%. There has been substantial progress in identification of new therapeutic targets, along with approval of at least three targeted therapies for AML in recent years. Nevertheless, treatment has largely remained unchanged over couple of decades, with ~40% patients not achieving remission. AML is a highly heterogenous disease and there is a need for a preclinical platform to understand the heterogeneity and tumor microenvironment that can guide therapy selection. In this study, we employed an ex vivo tumor explant model to study tumor microenvironment and to select a treatment course for AML patients. Our data reveal dysregulation of DNA methyltransferase (DNMT) and histone deacetylase (HDAC) in a subset of AML patients. Based on this observation, epigenetic modulators azacitidine and panobinostat alone and in combination, were evaluated as treatment regimens in cytarabine refractory tumors. More than 50% of the treated samples showed response to the combination therapy. In order to explore alternate treatment modalities for tumors refractory to these epigenetic modulators, TCGA data analysis was done which revealed increased expression and hypomethylation of IFNGR1/2, suggesting activation of JAK/STAT pathway in AML. This was further interrogated ex vivo, with p-STAT3 expression in patients' samples. Fedratinib, a JAK/STAT inhibitor was evaluated and 78% tumor efficacy response was achieved. Taken together, our data indicate that ex vivo platform derived from patient samples is capable in guiding optimal therapy selection for various classes of drugs including identification of novel targeted therapies.

Tumour heterogeneity is emerging as a key driver of drug response. Understanding the heterogeneity is therefore a critical milestone towards the prediction of clinical outcome of anti-cancer drugs. There is a need for technology that can predict treatment outcome with high fidelity by contextually integrating tumor heterogeneity and phenocopying the tumor microenvironment. Tumor grade-matched matrix support, activated immune compartments and autologous ligands from individual patients were used to engineer a novel personalized ex vivo platform named CANScript. We evaluated functional outcomes as a measure of response to a panel of anticancer drugs in this CANScript platform. Histopathological and molecular characterization of the tumor explants cultured in CANScript revealed a close approximation to the parental tumor at baseline as confirmed by tumor cell viability, proliferation, critical phosphoproteomic status, global transcriptomic profiles and balance in active components of tumor and stromal phenotypes (Majumder. B et al Nat. Commun, 2015). Using more than 100 patients clinical outcome of anticancer drugs (training set) we have further built a machine learning algorithm that takes all functional outcome data from the CANScript platform and provides outcome data in a linear scale showing clinical prediction. This algorithm when applied to the test cohort of more than 40 patients with refractory breast cancer assessed in the CANScript achieved more than 90% sensitivity while keeping specificity in a desired high range for predicting short term clinical outcome. The high specificity and sensitivity observed in predicting clinical outcomes using the CANScript support the use of this novel platform to select treatment for patients with breast cancer. Citation Format: Majumder PK, Majumder B, Mehrotra DG, Radhakrishnan P, Baraneedharan U, Agarwal S, Sengupta S, Babu G. Development and validation of personalized ex vivo platform mimicking patient heterogeneous tumor microenvironment to enable personalized treatment for breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-08-42.

Activation of the phosphoinositide 3-kinase pathway is commonly observed in human prostate cancer. Loss of function of phosphatase and tensin homolog (PTEN) is associated with the activation of AKT and mammalian target of rapamycin (mTOR) in many cancer cell lines as well as in other model systems. However, activation of mTOR is also dependent of kinases other than AKT. Here, we show that activation of mTOR is not dependent on AKT in a prostate-specific PTEN-deficient mouse model of prostate cancer. Pathway bifurcation of AKT and mTOR was noted in both mouse and human prostate tumors. We demonstrated for the first time that cotargeting mTOR and AKT with ridaforolimus/MK-8669 and M1K-2206, respectively, delivers additive antitumor effects in vivo when compared to single agents. Our preclinical data suggest that the combination of AKT and mTOR inhibitors might be more effective in treating prostate cancer patients than current treatment regimens or either treatment alone.

Background: The presence and activity of lymphocytes within the tumor is critical for clinical response to cancer immunotherapy, such as immune checkpoint blockade. Tumors with poor T-cell inflamed phenotypes, often referred to as a ‘cold' tumor, is associated with modest clinical response. High baseline infiltration of effector T-cell lymphocytes is considered ‘hot', and patients are predicted to respond more favorably to treatment. However, patient-to-patient response and durability remains highly variable. There is an urgent gap in available methods to study lymphocyte infiltration, trafficking and spatial heterogeneity induced by different cancer immunotherapies in individual patients. Moreover, there is a poor correlation between therapy-induced lymphocyte infiltration with clinical response, which could be shaped using personalized approaches to therapy. Methods: Here, we used CANscriptTM, an ex-vivo human tumor model that recapitulates and preserves the native, patient-autologous tumor microenvironment, including autologous patient-derived peripheral blood mononucleated cells (PBMC). Utilizing tissue from breast cancer patients classified as either ‘cold' or ‘hot', we studied lymphocyte infiltration under pressure of a-PD-1 immune checkpoint blockade (pembrolizumab) over a 72h time course. Using fluorescent labelling and flow cytometric analysis we characterized infiltrating lymphocytes, studying the role of T-cell repertoires under different environmental and immunotherapy pressures. We coupled these analyses with multiplex immunohistochemistry (CD3+, CD4+, CD8+, pan-cytokeratin, DAPI) to map spatial heterogeneity of tumor cells and lymphocytes before and after treatment, ex-vivo. Results: We determined that immune checkpoint blockade induced unique patterns of migration and infiltration of effector T-cells (Teff) and T-regulatory (Treg) cells in ‘hot' vs ‘cold' tumors. Furthermore, we determined that, in some instances, ‘cold' tumors can be driven towards a ‘hot' phenotype characterized by trafficking of active immune lymphocytes following treatment, which corresponded to differential ratio of Teff to Treg compared to baseline. Concluding remarks: Taken together, these data demonstrate the utility of CANscriptTM as a platform to characterize response to immunotherapy in a spatial context, providing insight into the migratory patterns of immune cell subsets at the individual patient level. Such an advance in our preclinical methods to study immuno-modulators may help guide treatment decisions for clinicians while simultaneously functioning as a platform to study and discover mechanisms of clinical efficacy for emerging drug combinations. Citation Format: Munisha Smalley, Basavaraja Shanthappa, Hans Gertje, Mark Lawson, Baraneedharan Ulaganathan, Allen Thayakumar, D.C. Doval, Anurag Mehta, S. P. Somashekhar, Padhma Radhakrishnan, Pradip Majumder, Aaron J. Goldman. Nonuniform T-cell infiltration induced by PD-1 checkpoint blockade, ex vivo, predicts distinct clinical response [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 705.

e24246Background: CANScript (TM), an ex-vivo tumor model, uses tumor explants and autologous patient (pt) plasma and peripheral blood mononuclear cells to replicate the tumor microenvironment (ME) ...

e18042Background: PD-1 inhibition has shown promise in second-line, platinum (Pt) resistant HNSCC (e.g. KEYNOTE-055). To improve the outcome of PD-1 blockade, clinical trials are testing immunother...

Predicting clinical response to anticancer drugs remains a major challenge in cancer treatment. Emerging reports indicate that the tumour microenvironment and heterogeneity can limit the predictive power of current biomarker-guided strategies for chemotherapy. Here we report the engineering of personalized tumour ecosystems that contextually conserve the tumour heterogeneity, and phenocopy the tumour microenvironment using tumour explants maintained in defined tumour grade-matched matrix support and autologous patient serum. The functional response of tumour ecosystems, engineered from 109 patients, to anticancer drugs, together with the corresponding clinical outcomes, is used to train a machine learning algorithm; the learned model is then applied to predict the clinical response in an independent validation group of 55 patients, where we achieve 100% sensitivity in predictions while keeping specificity in a desired high range. The tumour ecosystem and algorithm, together termed the CANScript technology, can emerge as a powerful platform for enabling personalized medicine., Efficacy of anticancer treatments vary across patients, imposing a need for personalized approaches. Here the authors show that responsiveness to chemotherapy can be predicted using tumour explant cultures in a patient-matched microenvironment, coupled with a machine-learning algorithm.

The PI3K/AKT/mTOR pathway is an important signaling axis that is perturbed in majority of cancers. Biomarkers such as pS6RP, GLUT1, and tumor FDG uptake are being evaluated in patient stratification for mTOR pathway inhibitors. In the absence of a clear understanding of the underlying mechanisms in tumor signaling, the biomarker strategy for patient stratification is of limited use. Here, we show that no discernible correlation exists between FDG uptake and the corresponding Ki67, GLUT1, pS6RP expression in tumor biopsies from patients with head and neck cancer. Correlation between GLUT1 and pS6RP levels in tumors was observed but elevated pS6RP was noticed even in the absence of concomitant AKT activation, suggesting that other downstream molecules of PI3K/AKT and/or other pathways upstream of mTOR are active in these tumors. Using an ex vivo platform, we identified putative responders to rapamycin, an mTOR inhibitor in these tumors. However, rapamycin did not induce antitumor effect in the majority of tumors with activated mTOR, potentially attributable to the observation that rapamycin induces feedback activation of AKT. Accordingly, treatment of these tumors with an AKT inhibitor and rapamycin uniformly resulted in abrogation of mTOR inhibition-induced AKT activation in all tumors but failed to induce antitumor response in a subset. Phosphoproteomic profiling of tumors resistant to dual AKT/mTOR inhibitors revealed differential activation of multiple pathways involved in proliferation and survival. Collectively, our results suggest that, in addition to biomarker-based segregation, functional assessment of a patient's tumor before treatment with mTOR/AKT inhibitors may be useful for patient stratification. Cancer Res; 73(3); 1118–27. ©2013 AACR.

Neovascularization is considered as a major event during embryonic development, found to be deregulated in several pathological conditions including malignancies. Factors secreted by tumor cells trigger a cascade of events which in turn play a critical role in the initiation and sprouting of tumor-specific-sustained-vasculature subsequently led to dramatic expansion of primary tumors, invasion and metastasis. Increasing evidence suggested that modulation of tumor cell specific intrinsic mechanisms which are accountable for tumor vasculogenesis would have therapeutic benefits to the patients even at later stages of disease. Therefore screening of such potential anti-angiogenic agents necessitates engineering a system where tumor vasculogenic environment is well preserved at micro-architecture level close to that of patient's native settings. Such a platform developed by Mitra Biotech, CANScriptTM demonstrated a high level of correlation to clinical outcome for many chemotherapeutics as well as targeted drugs in multiple solid and hematological cancers. Our CANScriptTM platform was being effectively validated for the retention of vascular phenotypes along with their immune and stromal counterparts, and their associated signaling networks. When patient derived tumor specimens were cultured in a two chambers microplates with human umbilical vein endothelial cells’ (HUVECs) in the presence of autologous ligands and customized matrix support, the native mediators released from tumor cells instigated complete formation of mature sprouts within 24 hours compared to HUVEC cells alone. In this platform we also assessed the anti-tumor efficacy of a known anti-angiogenic agents. The response to VEGF inhibitor was in turn measured by functional parameters like tumor morphology, anti-proliferative and apoptotic effects in combination with anti-angiogenic activity for the human tumors enrolled in the study. Treatment with VEGF inhibitor alone resulted in the abrogation of angiogenic crosstalk and transmission of signals from tumor cells to vessels. In addition to capturing anti-tumor efficacy of drugs using this platform technology, we were also able to dissect and better understand intervening strategies targeting tumor angiogenesis and vasculogenesis mechanisms under ex vivo conditions recreating in vivo like milieu. Citation Format: Baraneedharan Ulaganathan, Allen Thayakumar B, Manoj Rajappa, Ayyappan Velu, Arun Prasath, Kamal Ameer, Nilesh Brijwani, Padhma Radhakrishnan, Biswanath Majumder, Pradip K. Majumder, Saravanan Thiyagarajan. Intrinsic factors from patient's tumor help neovascularization in an ex vivo platform. [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 3397.

Prostate cancer is one of the leading malignancies in adult males worldwide and existing therapies often result in treatment failure and emergence of castration resistant prostate cancer (CRPC). Outcome of some recent clinical trials highlighted the benefit of Docetaxel as a part of initial therapy in CRPC. However, a significant number of patients still remain chemoresistant. Context dependent response profiling using a clinically oriented functional platform would further improve this scenario. Here, we elucidated the role of different immune cells and type 1 and type 2 cytokines in improving Docetaxel sensitivity to prostatic carcinoma. We tested Docetaxel sensitivity in prostate tumors (n = 10) using CANScriptTM platform, a personalized next generation functional testing tool (Majumder B et al. Nat. Commun 2015, Goldman A et al. Nat. Commun. 2015). Of these 10 tumors, 4 were found to be sensitive to Docetaxel and 6 were insensitive. The immune profiling of these tumors suggested that despite preferential CD45, CD8 and CD4 levels in majority of the responders both responders and non responders display modest numbers at baseline. We also observed similar heterogeneity in CD69 (T cell activation marker) in these two groups. Altered expression of cytokines was observed in Docetaxel sensitive and resistant tumors upon their co-culture with peripheral blood mononuclear cells (PBMC), suggesting a defined role of these cytokines in Docetaxel response. We extended the study further by testing the efficacy of PD-1/ PD-L1 inhibitors in Docetaxel resistant tumors. Our results provide the basis for validation of immune phenotypes and cytokines as therapeutic response biomarkers in Docetaxel treated prostatic tumors. 1. Majumder B et al. Nat. Commun.6:6169 (2015). 2. Goldman A et al. Nat. Commun. 6:6139 (2015). Citation Format: Debapriya G. Mehrotra, Nilesh Brijwani, Biplab Tewary, Vasathkumar Sekar, Abhishek Krishnan, Dency D. Pinto, Muthusamy Oliyarasi, Biswanath Majumder, Saravanan Thiyagarajan, Padhma Radhakrishnan, Pradip K. Majumder. Immunomodulating cytokine profiling of docetaxel-treated human prostate cancer specimens. [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 5121.

This study was designed to determine the amplification and polymorphism of epidermal growth factor receptor (EGFR) gene in prostatic diseases like benign hypertrophy (BPH) and carcinoma (CaP). The EGFR gene was found to be amplified in grade IV BPH as compared to grade II BPH. Digestion of genomic DNA with MspI and HpaII revealed the presence of a 5kb band following southern blot analysis. This 5kb band was present in all the CaP cases and in one out of three BPH cases. It is possible that such a polymorphism is associated with the type or extent of tumor progression.

Predicting clinical response to anticancer drugs remains a major challenge in the treatment of cancer. Indeed, while biomarker-guided strategies for personalizing anticancer drugs have shown strong promise in certain cases, recent studies have shown that the tumor microenvironment and heterogeneity can limit the predictive power of biomarkers alone. Here we have engineered a personalized tumor ecosystems, termed CANScript™, that contextually conserve tumor heterogeneity and phenocopy the tumor ecosystem using thin tumor explants maintained in defined tumor grade-matched matrix support and autologous ligands from patients. We then demonstrated that the CANScript™ platform can be used to predict clinical response. Specifically, functional readouts obtained by exposing the CANScript™ ecosystems from more than 1100 patients to a panel of anticancer drugs, together with the corresponding clinical outcomes, were used to train a novel machine learning algorithm; the learned model was then applied to predict clinical response to anticancer drugs in a test group comprising of 900 new patients, where it achieved 100% sensitivity in its predictions while also keeping specificity in a desired high range. We have also observed that CANScript™ retains patient tumor immune environment which is important for clinical response of not only immunomodulators but other anti-cancer drugs. Here we report the effect of immunomodulators in refractory CRC tumors which remains difficult to treat. Data demonstrates that Anti-CTLA-4 has profound antitumor effect in refractory CRC tumors by increasing cytotoxic T-Cells. In this cohort of 16 patients’ tumors CANScript™ platform identifies 31% as responders (5/16) where conventional tumor sections culture model shows only 6% as (1/16) responders. Citation Format: Biswanath Majumder, Baraneedharan Ulaganathan, Allen Thayakumar, Saravanan Thiyagarajan, Nilesh Brijwani, Biplab Tewari, Basavaraja U. Santhappa, Padhma Radhakrishnan, Pradip K. Majumder. Identification of responders for Anti-CTLA4 in refractory colorectal cancers using CANScript™ platform. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1304. doi:10.1158/1538-7445.AM2015-1304

6029 Background: Predicting clinical response to anticancer drugs remains a major challenge in the management of cancer. Recent advances show that Tumor Micro Environment (TME) and heterogeneity im...

Histone deacetylases (HDAC) are deregulated in many human cancers and a few HDAC inhibitors (HDACi) have been shown to be effective in patients with rare leukemia. However, clinical responses are not very promising for many HDACi in solid cancers. There is an urgent need to develop best in class HDACi with higher therapeutic index in solid cancers. At present less than 10% of anti-cancer drugs entering phase 1 clinical trials are successful in getting market approval. There are many factors for this poor clinical success for oncology therapeutics, and one of them is the lack of robust preclinical model driving clinical development. Cancer is a very heterogeneous disease and many of the present preclinical models do not represent this heterogeneity. As a result, while a candidate drug may appear good in present preclinical models, they often fail to make the cut in clinical development. To circumvent this critical problem, we have developed a robust TE (Tumor Ecosystem) platform technology using patient tumors, autologous ligands, and immune compartments from the same patients.Our TE platform preserves patient tumor heterogeneity along with other functional and genomic markers and immunogenic cytokines. Efficacy of drugs exposed to the patient's tumor tissue in our TE platform is assessed by as many as 17 orthogonal assays within a week. The results from these assays are converted into a single predictive score called the “M-Score,” and it has been clinically validated using FDA approved drugs in > 1,800 patients across multiple solid and hematological cancers. Our data shows that TE platform predicts treatment outcome of FDA approved drugs in clinical setting with very high sensitivity and specificity. We have the opportunity to use this platform technology to identify cancer indications and appropriate combinations for MIT-1102, a novel HDACi that exhibited potent histone deacetylase inhibitory activity. Our data further indicates that the anti-tumor efficacy of MIT-1102 is superior to Vorinostat and comparable with Pracinostat (SB-939) while being more tolerable than Pracinostat. Results indicated the superiority of MIT-1102 when combined with SOCs in a subset of patients with gastric and pancreatic cancers. We are also using different omics to identify markers associated with response in those tumor types. MIT-1102 has the potential to be more effective in a defined patient population than other molecules in this class. In summary, we have identified a novel, potent HDACi with drug-like properties, and identified the correct cancer indications and most effective combinations using TE platform Technology. Citation Format: Mallikarjun Sundaram, Baraneedharan Ulaganathan, Muthu Dhandapani, Allen Thayakumar, Pragnashree Mukhopadhyay, Saravanan Thiyagarajan, Biswanath Majumder, Prasad Shivarudriah, H Jagadheshan, Ganesh Sambasivam, Steve Birnbuam, Padhma Radhakrishnan, Pradip K. Majumder. Application of patient tumors-derived tumor ecosystem platform for the development of novel HDAC inhibitor in solid cancers. [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 3736. doi:10.1158/1538-7445.AM2014-3736

RNA isolated from frozen human postmortem prostate tissue was evaluated for its utility in molecular biological studies based on RNA analysis. Our results on slot-blot analysis show the presence of receptor transcripts for androgen, epidermal growth factor and basic fibroblast growth factor in postmortem prostate tissue obtained 20–120 hrs after death. However, the RNA in these samples was found to be degraded as revealed by the absence of ribosomal bands on gel electrophoresis. AR mRNA was found to be present in one of the five samples when analysed by northern blotting.

Histone deacetylases (HDAC) have been shown to be involved in oncogenic transformation by mediating transcription factors. Although a few HDAC inhibitors (HDACi) have been shown to be effective in patients with rare leukemia, there is a need to develop best in class HDACi with higher therapeutic index in solid cancers. PAT-1102 is a novel HDACi that exhibited potent histone deacetylase inhibitory activity and growth inhibitory activity against a panel of cell lines in in vitro and in vivo models. Studies indicated that PAT-1102 mediates target modulation (histone hyperacetylation), p21 up regulation, angiogenesis inhibition, and induction of cell cycle arrest in these models. PAT-1102 possesses drug like properties including good aqueous solubility, oral absorption, metabolic stability and favorable pharmacokinetic profile in rodents. It did not show any hERG inhibitory activity, CYP liability and was well tolerated in tumor bearing mice. It had showed a dose dependent tumor growth inhibition of up to 72% in cell line based xenograft models of lung and colorectal cancer. Our data further indicated that the anti-tumor efficacy of PAT-1102 was superior to Vorinostat and comparable with Pracinostat (SB-939) while being more tolerable than Pracinostat. Although it has pan histone deacetylase properties, it showed preferential inhibition of HDAC-3 & HDAC-6. PAT-1102 efficacy was further assessed using “Oncoprint®” platform, a clinically relevant preclinical technology that has been demonstrated to have very high correlation to clinical outcome for chemotherapeutics as well as targeted drugs in multiple solid and hematological cancers (with overall sensitivity of 88% and specificity of 90% in N=500+ patients). Using Oncoprint®, PAT-1102’s efficacy was assessed in patient derived solid cancers (Pancreas, Gastric, Esophagus, Head & Neck, and Colorectal). Further we assessed the efficacy of PAT-1102 alone as well as in combination with various Standard-Of-Care drugs (SOCs) and compared the outcome with efficacy derived from the use of SOCs alone. Our study has been powered to include 75-100 patient derived refractory/recurrent tumors in each indication. Current results indicated the superiority of PAT-1102 when combined with SOCs in a subset of patients with solid cancers. Pre-clinical data shows that PAT-1102 has no toxic effect when given alone or in combination with SOC at efficacious doses . PAT-1102 has the potential to be more effective in a defined patient population than other molecules in this class. In summary, we have identified a novel, potent HDACi with drug like properties. Our data suggest PAT-1102 has the potential to be best in class HDACi when combined with SOC in solid cancers. Clinical development of PAT-1102 in solid cancers will be initiated in 2013. Citation Format: Padhma Radhakrishnan, Muthu Dhandapani, Baraneedharan Ulaganathan, Allen Thayakumar, Saravanan Thiyagarajan, Dency Pinto, D R. Vinod, Biswanath Majumder, Prasad Shivarudriaah, H Jagadheshan, Ganesh Sambasivam, Pradip K. Majumder. PAT-1102, a novel HDAC inhibitor exhibits potent anti-tumor efficacy in patient-derived refractory solid tumors. [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 2051. doi:10.1158/1538-7445.AM2013-2051

One of the formidable challenges in cancer therapeutics is developing and validating a highly effective preclinical testing platform. However, existing in vitro and in vivo testing platforms show limited success. In this context, we have developed Oncoprint®, a novel clinically relevant preclinical platform demonstrated to have very high correlation to clinical outcome for chemotherapeutics as well as targeted drugs in multiple solid and hematological cancers (with overall sensitivity of 88% and specificity of 90% in more than 500 patients). Oncoprint® is a rapid reproducible ex-vivo tumor explant system, developed to mimic native tumor microenvironment. By including autologous paracrine growth factors, cancer specific customized matrix support, autologous immune environment along with other growth promoting conditions, significant improvements were observed in viability, proliferation of tumors including retention of tumor/stroma, cancer phenotypes, integrity at micro-architecture level and maintenance of functional signaling network. Using this platform we assessed the anti-tumor efficacy of Fragmin (a low molecular weight heparin-LMWH) alone as well as in combination with various Standard-Of-Care drugs (SOCs) and compared the outcome with efficacy derived from the use of SOCs alone in twenty patient derived pancreatic tumors. Clinical trial data indicates that Fragmin when administered in patients with advanced pancreatic cancer decreased levels of circulating-tissue factor antigen and attenuated induction of cellular invasion in their blood. Oncoprint data indicates that Fragmin has potent anti-angiogenic activity as a single agent (in ∼ 77% tumor samples) and enhanced anti-proliferative and apoptotic effect in combination with SOC (Cisplatin and Gemcitabine) compared to SOC alone in ∼ 30% of these pancreatic tumors. . Our study has been powered to include 75-100 patient derived refractory pancreatic tumors for further evaluation. Detailed molecular profiling studies are underway to understand the mechanism of action of this drug to delineate the biology behind response in the responder sub-population. Citation Format: Mallikarjun Sundaram, Saravanan Thiyagarajan, Muthu Dhandapani, Nilesh Brijwani, Arun Prasath, Allen Thayakumar, Ameer Kamal, Padhma Radhakrishnan, Biswanath Majumder, Baraneedharan Ulaganathan, Pradip K. Majumder. Human tumor derived Oncoprint® platform predicts molecular mechanism of sensitivity and resistance to Fragmin in pancreatic cancer. [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 5072. doi:10.1158/1538-7445.AM2013-5072

The PI3K/AKT/mTOR pathway is an important signaling axis that is perturbed in majority of cancers. Biomarkers such as pS6RP, GLUT1 and tumor FDG uptake are being evaluated in patient stratification for mTOR pathway inhibitors. In the absence of a clear understanding of the underlying mechanisms in tumor signaling, the biomarker strategy for patient stratification is of limited use. Here, we show that no discernible correlation exists between FDG uptake and the corresponding Ki67, GLUT1, pS6RP expression in tumor biopsies from Head and Neck Cancer (HNC) patients. Correlation between GLUT1 and pS6RP levels in tumors was observed but elevated pS6RP was noticed even in the absence of concomitant AKT activation, suggesting other downstream molecules of PI3K/AKT and/or other pathways upstream of mTOR, are active in these tumors. Using an ex-vivo platform, we identified putative responders to Rapamycin, an mTOR inhibitor in these tumors. However, Rapamycin did not induce anti-tumor effect in the majority of tumors with activated mTOR, potentially attributable to the observation that Rapamycin induces feedback activation of AKT. Accordingly, treatment of these tumors with an AKT inhibitor and Rapamycin uniformly resulted in abrogation of mTOR inhibition induced AKT activation in all tumors but failed to induce anti-tumor response in a subset. Phosphoproteomic profiling of tumors resistant to dual AKT/mTOR inhibitors revealed differential activation of multiple pathways involved in proliferation and survival. Collectively, our data suggest that in addition to biomarker based segregation, functional assessment of patient tumor prior to treatment with mTOR/AKT inhibitors might be useful for patient stratification. Citation Format: Padhma Radhakrishnan, Ulaganathan Baraneedharan, Muthu Dhandapani, Allen Thyakumar, Dency Pinto, Arun Prasath, Ayyappan Velu, A Kamal, Nilesh Brijwani, Rohini Nair, R Basavaraja, Misti Jain, Saravanan Thiyagarajan, Biswanath Majumder, Mallik Sundaram, Prasad Narayanan, Vikram D. Kekatpure, Pradip K. Majumder. Inhibition of Rapamycin induced AKT activation elicits differential anti-tumor response in head and neck cancers. [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 4947. doi:10.1158/1538-7445.AM2013-4947

Colorectal cancer (CRC) continues to have a dismal prognosis (overall five year survival is 11%). Majority of patients with CRC are either over-treated or under-treated as there is no technology in use for predicting treatment outcome. Currently, monoclonal antibodies that target EGFR have been approved for the treatment of metastatic CRC. However, only 10% to 20% of patients with metastatic CRC clinically benefit from anti-EGFR therapy. This indicates that molecular alterations other than EGFR are associated with their response. The aim of the present study is to use Oncoprint- our integrated platform technology that combines functional assays with molecular profiling to predict the clinical outcome to anti-cancer agents including anti-EGFR therapies. Forty tumors were obtained from patients with primary CRC and local metastasis with due informed consent. Tumors were sectioned and cultured in customized 96 well plates coated with CRC specific extracellular matrix (ECM) in the presence of their respective autologous ligands and were treated with anti-EGFR agent. Pharmacodynamic analysis of anti-EGFR agents was determined by phospho-EGFR and phospho-ERK immunohistochemistry. Tumor cell viability was assessed by WST, cellular proliferation was measured by Ki-67 and cell death was assessed by TUNEL and activated caspase-3. Tumor cell content and tumor/stroma ratio was evaluated using histopathological analysis. Mutations of KRAS/BRAF pathway and global microarray data were used to understand the molecular basis of response and non-response to anti-EGFR in these patient tumors. Functional read-out from Oncoprint platform showed that 30% of these tumors are responsive to anti-EGFR treatment. Molecular profiling of these tumors showed that they were wild type for K-RAS and B-RAF. Microarray data also suggest that RAS gene signature is a predictor of response. Mutation of either KRAS (codon 12/13/61/146) or BRAF (V600E) confers non-response to anti-EGFR treatment. However, wild type KRAS/BRAF was observed in ∼40% of non-responders. RAS gene signature was conspicuously absent in these patients. Clinical follow-up of these patients is under progress. Collectively these findings indicate that there are factors other than K-RAS mutation that may be indicative of the true response or non-response of anti-EGFR treatments under clinical setting. Oncoprint platform may be able to predict the true clinical response of anti cancer therapies including anti-EGFR treatments. Further, molecular profiling of individual tumor along with the efficacy analyses by Oncoprint would be beneficial for the identification of driver mutations and the selection of appropriate treatment options for these patients. 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 3191. doi:1538-7445.AM2012-3191

Prostate cancer is the third most common cause of cancer-related deaths in men and is most prevalent in developed countries. The androgen signaling pathway plays a critical role in the development and maintenance of the prostate gland and is hyper-activated in prostate cancer. Additionally, gain of function mutations in the PI3 kinase pathway is associated with progression of this disease. Using the prostate-specific Pten-deleted mouse model of cancer, we show that these two pathways are distinct for tumor growth and disease progression. Depletion of androgens by either surgical or chemical castration significantly inhibits tumor growth without altering the activation of Akt and mTOR. We demonstrate that inhibition of mTOR with ridaforolimus or inhibition of Akt with MK-2206, combined with androgen deprivation (bicalutamide), results in additive anti-tumor effects in vivo when compared to single agent treatment. In this model, we also demonstrate that Akt inhibition does not block mTOR kinase activation, and that inhibiting both Akt and mTOR kinases together significantly enhances anti-tumor efficacy. Our data suggest that the combination of an anti-androgen with an mTOR inhibitor and/or Akt inhibitor may be more effective than standard therapies in treating androgen-dependent prostate cancer patients with an activated PI3 kinase pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-284.

c-Met and ErbB family of receptors including EGFR, ErbB-2, ErbB-3 and ErbB-4 are co-expressed in many human tumors including Non-Small Cell Lung Cancers (NSCLC). Several reports suggest that the activation of c-Met and ErbB family receptors is associated with poor prognosis in multiple tumors types. Recently it has been shown that activation of c-Met is common in Erlotinib and Gefitinib-resistant NSCLC patients. Combining c-Met and EGFR inhibitors has been proposed as a strategy for treatment in EGFR inhibitor-refractory lung cancers. To understand the cooperation of these pathways, we used a c-Met activated human NSCLC cell line (EBC-1), where EGFR signaling network is also active. In vitro treatment of EBC-1 cells with a combination of Erlotinib and MK-8033 (an inhibitor of c-Met and RON kinases) resulted in greater growth inhibition relative to treatment with either agent alone. In order to validate these in vitro data, we have tested MK-8033 and EGFR inhibitors (Erlotinib and Gefitinib) in vivo. The data suggests the effect of MK-8033 with Gefitinib or Erlotinib on tumor growth is greater when combined as compared to monotherapy in this preclinical xenograft model. PK data suggests no drug-drug interaction of MK-8033 and EGFR inhibitor when administered together. Exposure of these agents was sufficient to inhibit both kinases in tumors. Our studies provide a rationale for the combination of MK-8033 and Erlotinib or Gefitinib in EGFRi refractory patients with NSCLC. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C209.

Gastric cancer is currently the second most frequent cancer-related cause of death with an incidence rate of approximately one million cases annually. The present worldwide standard of care treatment regimen for gastric cancer is the combination of 5-fluorouracil (5-FU) and platinum analog (cisplatin). Many receptor tyrosine kinases are deregulated in gastric cancer including erbB2, c-Met, and EGFR. The purpose of our study was to dissect the role of c-Met signaling in gastric cancer and test the effects of our c-Met inhibitor (MK-8033) alone and in combination with the standard of care agents 5-FU and cisplatin. As a monotherapy, MK-8033 potently inhibited proliferation in a panel of gastric cancer cell lines in vitro. When administered simultaneously with cisplatin and 5-FU, the effects of the combination were diverse, ranging from additive to antagonistic. Altering the treatment schedule by the addition of 5-FU and cisplatin prior to MK-8033 resulted in an overall combination benefit across the panel. In a gastric cancer xenograft model, while MK-8033 alone significantly inhibited tumor growth progression, the co-administration of 5-FU and cisplatin with MK-8033 showed greater benefit than either the monotherapy or the standard regimen. In vitro cell cycle and survival analysis support the observed in vivo data, and provides a mechanistic rationale for the observed combination benefit. Our data suggest the combination of c-Met inhibitors with standard of care agents 5-FU and cisplatin may be beneficial in gastric cancer patients with activated c-Met signaling. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C204.