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Supplementary Data from MK-2206, an Allosteric Akt Inhibitor, Enhances Antitumor Efficacy by Standard Chemotherapeutic Agents or Molecular Targeted Drugs In vitro and In vivo

Supplementary Figure 1, Table 1 from De novo Discovery of a γ-Secretase Inhibitor Response Signature Using a Novel In vivo Breast Tumor Model

Supplementary Figures 1-4 from Inhibition of Tumor Growth Progression by Antiandrogens and mTOR Inhibitor in a Pten-Deficient Mouse Model of Prostate Cancer

Supplementary Figure S1 from Androgen-Induced Differentiation and Tumorigenicity of Human Prostate Epithelial Cells

Supplementary Figures 1-3, Tables 1-2 from Downregulation of Notch Pathway by a γ-Secretase Inhibitor Attenuates AKT/Mammalian Target of Rapamycin Signaling and Glucose Uptake in an ERBB2 Transgenic Breast Cancer Model

Supplementary Tables S1 & S2 from Androgen-Induced Differentiation and Tumorigenicity of Human Prostate Epithelial Cells

Supplementary Methods and Table S3 from Androgen-Induced Differentiation and Tumorigenicity of Human Prostate Epithelial Cells

Notch pathway signaling plays a fundamental role in normal biological processes and is frequently deregulated in many cancers. Although several hypotheses regarding cancer subpopulations most likely to respond to therapies targeting the Notch pathway have been proposed, clinical utility of these predictive markers has not been shown. To understand the molecular basis of γ-secretase inhibitor (GSI) sensitivity in breast cancer, we undertook an unbiased, de novo responder identification study using a novel genetically engineered in vivo breast cancer model. We show that tumors arising from this model are heterogeneous on the levels of gene expression, histopathology, growth rate, expression of Notch pathway markers, and response to GSI treatment. In addition, GSI treatment of this model was associated with inhibition of Hes1 and proliferation markers, indicating that GSI treatment inhibits Notch signaling. We then identified a pretreatment gene expression signature comprising 768 genes that is significantly associated with in vivo GSI efficacy across 99 tumor lines. Pathway analysis showed that the GSI responder signature is enriched for Notch pathway components and inflammation/immune-related genes. These data show the power of this novel in vivo model system for the discovery of biomarkers predictive of response to targeted therapies, and provide a basis for the identification of human breast cancers most likely to be sensitive to GSI treatment. [Cancer Res 2009;69(23):8949–57]

Androgen ablation is the primary treatment modality for patients with metastatic prostate cancer; however, the role of androgen receptor signaling in prostate cancer development remains enigmatic. Using a series of genetically defined immortalized and tumorigenic human prostate epithelial cells, we found that introduction of the androgen receptor induced differentiation of transformed prostate epithelial cells to a luminal phenotype reminiscent of organ-confined prostate cancer when placed in the prostate microenvironment. Moreover, androgen receptor expression converted previously androgen-independent, tumorigenic prostate epithelial cells into cells dependent on testosterone for tumor formation. These observations indicate that androgen receptor expression is oncogenic and addictive for the human prostate epithelium.

Cancer progresses by evading the immune system. Elucidating diverse immune evasion strategies is a critical step in the search for next-generation immunotherapies for cancer. Here we report that cancer cells can hijack the mitochondria from immune cells via physical nanotubes. Mitochondria are essential for metabolism and activation of immune cells. By using field-emission scanning electron microscopy, fluorophore-tagged mitochondrial transfer tracing and metabolic quantification, we demonstrate that the nanotube-mediated transfer of mitochondria from immune cells to cancer cells metabolically empowers the cancer cells and depletes the immune cells. Inhibiting the nanotube assembly machinery significantly reduced mitochondrial transfer and prevented the depletion of immune cells. Combining a farnesyltransferase and geranylgeranyltransferase 1 inhibitor, namely, L-778123, which partially inhibited nanotube formation and mitochondrial transfer, with a programmed cell death protein 1 immune checkpoint inhibitor improved the antitumour outcomes in an aggressive immunocompetent breast cancer model. Nanotube-mediated mitochondrial hijacking can emerge as a novel target for developing next-generation immunotherapy agents for cancer. Cancer cells adopt a series of strategies to evade the immune response mounted by the organism against them. Here we find that tumour cells can hijack mitochondria from immune cells by forming physical nanotubes, and suggest that inhibiting this process might represent a potential immunotherapy approach.

Vasculogenic mimicry (VM), defined as an endothelial cell independent alternative mechanism of blood and nutrient supply by dysregulated tumor cells, is associated with poor prognosis in oral squamous cell carcinoma (OSCC). Here we aim to investigate the underlying molecular mechanism of the synergistic effect of phytochemical Lupeol and standard microtubule inhibitor Paclitaxel in reversing the hypoxia induced VM formation in OSCC. The results demonstrated that the hypoxia induced upregulation of HIF-1α led to augmentation of signaling cascade associated with extracellular matrix remodeling and EMT phenotypes that are mechanistically linked to VM. Induction of HIF-1α altered the expression of EMT/CSC markers (E-Cadherin, Vimentin, Snail, Twist and CD133) and enhanced the ability of cell migration/invasion and spheroid formation. Subsequently, the targeted knockdown of HIF-1α by siRNA led to the perturbation of matrigel mediated tube formation as well as of Laminin-5γ2 expression with the down-regulation of VE-Cadherin, total and phosphorylated (S-897) EphA2, pERK1/2 and MMP2. We also observed that Lupeol in association with Paclitaxel resulted to apoptosis and the disruption of VM associated phenotypes in vitro. We further validated the impact of this novel interventional approach in a patient derived tumor explant culture model of oral malignancy. The ex vivo tumor model mimicked the in vitro anti-VM potential of Lupeol-Paclitaxel combination through down-regulating HIF-1α/EphA2/Laminin-5γ2 cascade. Together, our findings elucidated mechanistic underpinning of hypoxia induced Laminin-5γ2 driven VM formation highlighting that Lupeol-Paclitaxel combination may serve as novel therapeutic intervention in perturbation of VM in human OSCC.

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.

Background: Immune checkpoint inhibitors are being used in clinics for a diverse set of cancers. However, the outcome is highly contextual and personalized with consistently low response rates. Understanding the mechanistic barriers at single cell levels is key for novel candidate selection. Here we describe Dexter࣪ microfluidic platform that can create highly parallel nanobioreactors on a chip to study cellular functions and interactions at the single cell level. Using microliter volumes of live tumor and human PBMC, we showed modulation of dynamic tumor-immune crosstalk at single cell level. Methods: The Dexter࣪ microfluidic platform was used for functional characterization at single cell resolution. The platform integrates an array of nanoliter sized droplets with cell encapsulation, multicolor fluorescence imaging and selective cell retrieval. PBMC, RBC and cancer cells were fluorescently (CFSE, PKH) labelled and used to interrogate effector cell functions following exposure to immune modulating milieu in nanoliter droplets. CD8+T cells were activated and labelled. Individual and clustered tumor cells were challenged with stimulated PBMC. Finally, using bioinformatic tools, we construct a Tumor immune Interactive Network (TiIN) and simulate perturbation. Results: Our data confirmed preservation of surface markers, T cells and tumor-immune specific intracellular and surface events including cell-cell contacts at the level observed before their encapsulation in microfluidic droplets. Both size and intensity of the signals were maintained without significant loss. Platform displayed critical events like antigen presentation, tumor-probiotic interface, T cell engagement with microbeads similar to APC dimensionalities and function. Both spiked tumor and immune cells showed high degree of recovery. Spatially resolved dynamic profiling in this platform further revealed immune phenotypic plasticity and vulnerabilities of tumor cells upon exposure to immune checkpoint agents. Finally, we, measured the killing of tumor cells by using different effector : target ratio that complement hot and cold niche. We observed differential killing of target when T cells were primed using immunomodulators. Conclusion: Our data highlights the versatility of Dexter࣪ platform in deciphering mechanistically critical tumor-immune interactions while parallelly resolving single cell events. This offers suitable means of profiling response to novel immunooncology candidates. Citation Format: Ashwin Lal, Ranjeet Singh, Abhay Sane, Rushil Manglik, Ravi R. Keshari, SM Mohanasundaram, Pradip K. Majumder, Biswanath Majumder. Dexter࣪ microfluidic platform spatially resolves antigen presentation and T cell functions in microdroplets and deciphers dynamic tumor-immune interaction network [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5383.

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.

Summary Ex vivo human tumor models have emerged as promising, yet complex tools to study cancer immunotherapy response dynamics. Here, we present a strategy that integrates empirical data from an ex vivo human system with computational models to interpret the response dynamics of a clinically prescribed PD-1 inhibitor, nivolumab, in head and neck squamous cell carcinoma (HNSCC) biopsies (N = 50). Using biological assays, we show that drug-induced variance stratifies samples by T helper type 1 (Th1)-related pathways. We then built a systems biology network and mathematical framework of local and global sensitivity analyses to simulate and estimate antitumor phenotypes, which implicate a dynamic role for the induction of Th1-related cytokines and T cell proliferation patterns. Together, we describe a multi-disciplinary strategy to analyze and interpret the response dynamics of PD-1 blockade using heterogeneous ex vivo data and in silico simulations, which could provide researchers a powerful toolset to interrogate immune checkpoint inhibitors., Graphical Abstract, Highlights • Computational strategy to study anticancer immune checkpoint blockade, ex vivo • PD-1 blockade-induced T helper type 1 (Th1) stratifies tumor biopsies, ex vivo • Systems biology links drug effect to dynamic intratumor T cell proliferation • In silico sensitivity analyses of PD-1 blockade predict Th1-induced antitumor effects, Biological Sciences; Cancer Systems Biology; Immunology; Systems Biology

BackgroundImmunotherapies in recent years emerged as effective treatment modalities for primary and metastatic tumors. However, a durable response besides a high objective response rate (ORR) is still elusive. Limitations of predictive functional platforms in providing spatial and temporal mechanistic insights into tumors and their dynamic immune proximity at the single-cell level critically highlight the challenges of modeling successful therapy outcomes. Here we describe the development of a novel microfluidic platform integrated with AI that incorporates single-cell sorting and functional interactions between different immune cells and tumors.MethodsThe DexterTM microfluidic platform involves the generation of a large number of nanoliter droplets (> 10,000) and mechanical dispensing of sorted droplets in a spatially defined array of nano-wells and achieves high throughput (> 1,000) independent nano bioreactors. Its open design concept illustrates simple workflow, easy perfusion through a gas permeable oil, sufficient nutrient availability, and offers a protective cushion around the cell for minimizing any impending stress during the downstream process. Indeed, the droplets in the array are spatially segregated to avoid any cross-talk. The platform is integrated with a multicolor fluorescence and bright field imager equipped with a culture stage and precise motion robotic arm in proximity to the droplet of interest for its efficient aspiration and release into mapped well.ResultsTo demonstrate the applications of this platform for functional modulation, we used differentially labeled immune cells like CD45, CD4, CD8 and detected cytokines like IFNγ and granzyme B at single-cell level; antigen uptake by dextran-FITC endocytosis. We further quantified the functionalities critical for immune-immune interactions, tumor-immune synapse formation, and killing of target tumor cells in a dynamic PBMC and tumor (breast, colon, and head and neck cancer cells) coculture setting. Longitudinal imaging showed the ability of the platform to capture the dynamic state of the antigen uptake and presentation, functional activation of driver phenotypes like CD8+cytotoxic T cells, and subsequent directed killing of tumor cells upon reinvigoration of effector phenotypes. Further coupling of AI-guided matrix with features from polyfunctional modulation accurately demonstrated the mechanistic shift choreographed upon immune activation.ConclusionsTogether, these findings illustrate the preclinical relevancy of a therapy agonist multimodality correlative platform integrating a controllable microfluid system and AI for real-time tracking of dynamic tumor-immune interplay at the level of single-cell resolution. Further validation of this system will offer opportunities for identifying driver modulation linked to predicted therapy response and modeling next-generation cell-based immunotherapies.

Cancer progresses by evading the immune system. Elucidating diverse immune evasion strategies is a critical step in the search for next-generation immunotherapies for cancer. Here we report that cancer cells can hijack the mitochondria from immune cells via physical nanotubes. Mitochondria are essential for metabolism and activation of immune cells. By using field-emission scanning electron microscopy, fluorophore-tagged mitochondrial transfer tracing and metabolic quantification, we demonstrate that the nanotube-mediated transfer of mitochondria from immune cells to cancer cells metabolically empowers the cancer cells and depletes the immune cells. Inhibiting the nanotube assembly machinery significantly reduced mitochondrial transfer and prevented the depletion of immune cells. Combining a farnesyltransferase and geranylgeranyltransferase 1 inhibitor, namely, L-778123, which partially inhibited nanotube formation and mitochondrial transfer, with a programmed cell death protein 1 immune checkpoint inhibitor improved the antitumour outcomes in an aggressive immunocompetent breast cancer model. Nanotube-mediated mitochondrial hijacking can emerge as a novel target for developing next-generation immunotherapy agents for cancer.

The tumor suppressor protein p53 is known to control cell cycle arrest and apoptosis. Lupeol is a phytochemical that has been found to induce apoptosis in different cancer types through the extrinsic pathway. As yet, however, its role in the induction of cell cycle arrest and apoptosis through the intrinsic pathway in head and neck cancer has not been investigated. Here, we aimed at understanding the mechanism underlying the antitumor effect of Lupeol in head and neck cancer. The antitumor effect of Lupeol on oral and laryngeal carcinomas was assessed using two in vitro 2D cell line models (HEp-2, UPCI:SCC-131) and, subsequently, an ex vivo 3D tumor explant culture platform that maintains key features of the native tumor microenvironment. The mechanism underlying Lupeol-mediated antitumor responses was delineated using MTT, colony formation, flow cytometry, immunofluorescence, Western blotting and immunohistochemistry assays. We found that Lupeol induced an enhanced expression of p53 in both cell line models tested and, subsequently, cell cycle arrest at the G1 phase. In addition we found that, following Lupeol treatment, p53 induced Bax expression and activated the intrinsic apoptotic pathway (as measured by Caspase-3 cleavage). Interestingly, Lupeol was also found to trigger G1 cell cycle arrest through up-regulation of the expression of CDKN2A, but not p21, resulting in inhibition of CyclinD1. In an ex vivo platform Lupeol was found to impart a potent antitumor response as defined by inhibition of Ki67 expression, decreased cell viability and concomitant activation (cleavage) of Caspase-3. Finally, we found that Lupeol can re-sensitize primary head and neck squamous cell carcinoma (HNSCC) tumor samples that had clinically progressed under a Cisplatin treatment regimen. Together, our data indicate that Lupeol may orchestrate a bifurcated regulation of neoplastic growth and apoptosis in head and neck cancers and may serve as a promising agent for the management of tumors that have progressed on a platinum-based treatment regimen.

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.

Background: Combined cancer immunotherapy is an emerging paradigm for the treatment of cancer. PD-L1, the cognate receptor for the programmed death receptor 1 (PD-1) is expressed in up to 40% of gastric/gastroesophageal junction (GEJ) tumors. Despite this, patients show modest responses to PD-1 inhibitors. A recent clinical trial, Checkmate-032, demonstrated superior efficacy when PD-1 inhibitor, Nivolumab, was combined with CTLA-4 inhibitor, Ipilimumab. Despite this, biomarkers that predict clinical response remain poorly understood. Methods: Here, with informed consent under IRB, fresh tumor biopsies were obtained from a randomly-selected cohort of gastric/GEJ patients (N=39). We then employed CANscriptTM, a clinically-validated human autologous ex-vivo tumor platform to interrogate the pharmacodynamics and prognostic efficacy (M-Score) of nivolumab +/- ipilimumab. PDL-1 status was determined for every patient by immunohistochemistry, flow cytometry was performed to analyze intratumor CD4+ and CD8+ T-cell profiles before and after treatment, and RNA seq. gene expression was performed to understand mechanistic changes under drug pressure. Results: We predicted, based on M-Score, that single agent nivolumab results in an overall response rate (ORR) of 10%, which closely matched previous single agent results from Checkmate-032 (10%). In contrast, we determined the combination of ipi+nivo resulted in a response rate of 20%, a finding consistent with reported clinical data (Checkmate-032; ORR 18%). Interestingly, we determined that only 28% of patients who responded to the ipi+nivo combination were predicted to respond to the monotherapy, suggesting the increased efficacy of the combination. RNA seq revealed unique features of adaptive immunity in the subset of patients who were predicted to respond to the combination immunotherapy, while PD-L1 status was a less-important biomarker to predict response. Conclusions: These results demonstrate the utility of the CANscript system to profile clinical response of immunotherapy. Our efforts may better inform clinical trial design and patient stratification before therapy to improve the potential for response. Citation Format: Vidushi Kapoor, Munisha Smalley, Nandini Pal Basak, Abhishek Basu, Manjusha Biswas, Manas Kumar Mandal, Pinaki Roy, Pradip K. Majumder, Aaron Goldman. Patient-level pharmacodynamics of response to combined ipilimumab and nivolumab for gastric cancer using a human autologous ex-vivo platform, CANscript [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 3212.

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.

Human breast tumors comprise a minor sub-population of tumor-initiating cells (TICs), commonly termed cancer stem cells. TICs are thought to sustain tumor growth and to confer resistance to current anticancer therapies. Hence, targeting TIC may be essential to achieving durable cancer cures. To identify molecular targets in breast TIC, we employed a transgenic mouse model of ERBB2 breast cancer; tumors arising in this model comprise a very high frequency of TIC, which is maintained in tumor cell populations propagated in vitro as non-adherent tumorspheres. The Notch pathway is dysregulated in human breast tumors and overexpression of constitutively active Notch proteins induces mammary tumors in mice. The Notch pathway has also been implicated in stem cell processes including those of mammary epithelial stem cells. Hence, we investigated the potential that the Notch pathway is required for TIC activity. We found that an antagonist of Notch signaling, a gamma (γ)-secretase inhibitor termed MRK-003, inhibited the survival of tumorsphere-derived cells in vitro and eliminated TIC as assessed by cell transplantation into syngeneic mice. Whereas MRK-003 also inhibited the self-renewal and/or proliferation of mammosphere-resident cells, this effect of the inhibitor was reversible thus suggesting that it did not compromise the survival of these cells. MRK-003 administration to tumor-bearing mice eliminated tumor-resident TIC and resulted in rapid and durable tumor regression. MRK-003 inhibited the proliferation of tumor cells, and induced their apoptosis and differentiation. These findings suggest that MRK-003 targets breast TIC and illustrate that eradicating these cells in breast tumors ensures long-term, recurrence-free survival.

SummaryActivation of the PI3K-AKT pathway in tumors is modulated by negative feedback, including mTORC1-mediated inhibition of upstream signaling. We now show that AKT inhibition induces the expression and phosphorylation of multiple receptor tyrosine kinases (RTKs). In a wide spectrum of tumor types, inhibition of AKT induces a conserved set of RTKs, including HER3, IGF-1R, and insulin receptor. This is in part due to mTORC1 inhibition and in part secondary to a FOXO-dependent activation of receptor expression. PI3K-AKT inhibitors relieve this feedback and activate RTK signaling; this may attenuate their antitumor activity. Consistent with this model, we find that, in tumors in which AKT suppresses HER3 expression, combined inhibition of AKT and HER kinase activity is more effective than either alone.

Background: Phenotypic cell state transitions are emerging as novel drivers of transient resistance to cancer chemotherapy. We recently demonstrated that non-cancer stem cells are able to undergo a phenotypic cell state transition that enables acquire a ‘reversible' drug tolerant state. Consequently, we made two key discoveries, drug tolerance 1) results in a cross-resistance to other classes of anticancer drugs 2) coincidental to a switch in the metabolic behavior. Further, evidences prompted to investigate if drug-induced metabolic reprogramming contributes to combination therapy resistance in a population of cancer cells that have gained tolerance to a primary therapy. Methods: We investigated the metabolic phenotype of drug tolerant cells using 3-D in-vitro models of breast cancer. A systems biology approach was used to identify key, interconnecting proteins involved in metabolic dysregulation, drawing inferences among signaling networks and events in a temporal context. Using an in-silico simulation we perturbed glucose metabolism, and tested how timing, combination and drug order impact the therapeutic effect of combination therapy and validated our findings using in-vivo experiments. Finally, to provide a direct clinical translation, we tested temporally-sequenced 3-drug combinations using CANscriptTM, a human explant tumor assay that captures the entire tumor ecosystem. Results: We report that conventional chemotherapies used to treat breast cancer, results in an adaptive cross-tolerance against an unrelated chemotherapeutic agent via induction of both glycolytic and oxidative pathways. These drug-tolerant cells switch to a CD44Hi phenotypic cell state, and rely on both the Akt pathway and HIF1α-Glut1 axis in a reactive oxygen species-dependent manner, which temporally cooperate to remodel a glucose shunt towards the pentose phosphate pathway. Mathematically modeling these pathways, we demonstrate how a sequentially-applied, 3-drug combination that includes G6PD metabolic inhibitors and cytotoxic agents can improve therapeutic effect. The use of CANscriptTM demonstrated that pharmacodynamics, biomarkers of resistance, and temporal ordering of drugs can influence the phenotypic response to therapy, reflecting in-vitro and in-vivo evidence, in a patient-specific manner. Conclusions: Timing, sequence and order of drugs is emerging as a critical component of combination therapy for cancer. Our results demonstrate that timing the order of G6PD inhibitors in exquisitely sequenced combination with chemotherapy can emerge as a new paradigm in the treatment of cancer. Ex-vivo, human tumor models that fully capture the tumor microenvironment can contribute to and potentially uncover the mechanisms of action, phenotypic effect, and pharmacodynamics of anticancer drug combinations in distinct temporal sequences. Citation Format: Baraneedharan Ulaganathan, Andrew Dhawan, Biswanath Majumder, Munisha Smalley, Saravanan Thiyagarajan, Gopinath S. Kodaganur, S Krishnamurthy, Mohammed Mamunur Rahman, Elizaveta Freinkman, Pradip Majumder, Mohammad Kohandel, Aaron Goldman. Temporal sequencing of anticancer drugs, ex vivo, optimizes therapeutic effect by targeting drug-induced glucose-6-phosphate dehydrogenase [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 2835.

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...

ERBB2/neu and Notch signaling are known to be deregulated in many human cancers. However, pathway cross-talk and dependencies are not well understood. In this study, we use an ERBB2-transgenic mouse model of breast cancer (neuT) to show that Notch signaling plays a critical role in tumor maintenance. Inhibition of the Notch pathway with a γ-secretase inhibitor (GSI) decreased both the Notch and the mammalian target of rapamycin/AKT pathways. Antitumor activity resulting from GSI treatment was associated with decreased cell proliferation as measured by Ki67 and decreased expression of glucose transporter Glut1. Positron emission tomography (PET) imaging showed that the functional consequences of decreased Glut1 translated to reduced glucose uptake and correlated with antitumor effects as measured by micro-computed tomography imaging. The decrease of Glut1 in neuT tumors was also observed in several human breast cancer cell lines following GSI treatment. We provide evidence that ∼27% of ERBB2-positive human breast cancer specimens display high expression of HES1, phospho-S6RP, and GLUT1. Together, these results suggest that pathways downstream of Notch signaling are, at least in part, responsible for promoting tumor growth in neuT and also active in both neuT and a subset of human breast cancers. These findings suggest that GSI may provide therapeutic benefit to a subset of ERBB2-positive breast cancers and that [18F]FDG-PET imaging may be useful in monitoring clinical response. Cancer Res; 70(6); 2476–84

Notch pathway signaling plays a fundamental role in normal biological processes and is frequently deregulated in many cancers. Although several hypotheses regarding cancer subpopulations most likely to respond to therapies targeting the Notch pathway have been proposed, clinical utility of these predictive markers has not been shown. To understand the molecular basis of γ-secretase inhibitor (GSI) sensitivity in breast cancer, we undertook an unbiased, de novo responder identification study using a novel genetically engineered in vivo breast cancer model. We show that tumors arising from this model are heterogeneous on the levels of gene expression, histopathology, growth rate, expression of Notch pathway markers, and response to GSI treatment. In addition, GSI treatment of this model was associated with inhibition of Hes1 and proliferation markers, indicating that GSI treatment inhibits Notch signaling. We then identified a pretreatment gene expression signature comprising 768 genes that is significantly associated with in vivo GSI efficacy across 99 tumor lines. Pathway analysis showed that the GSI responder signature is enriched for Notch pathway components and inflammation/immune-related genes. These data show the power of this novel in vivo model system for the discovery of biomarkers predictive of response to targeted therapies, and provide a basis for the identification of human breast cancers most likely to be sensitive to GSI treatment. [Cancer Res 2009;69(23):8949–57]

Background and purpose: γ-Secretase inhibitors (GSIs) block NOTCH receptor cleavage and pathway activation and have been under clinical evaluation for the treatment of malignancies such as T-cell acute lymphoblastic leukaemia (T-ALL). The ability of GSIs to decrease T-ALL cell viability in vitro is a slow process requiring >8 days, however, such treatment durations are not well tolerated in vivo. Here we study GSI's effect on tumour and normal cellular processes to optimize dosing regimens for anti-tumour efficacy. Experimental approach: Inhibition of the Notch pathway in mouse intestinal epithelium was used to evaluate the effect of GSIs and guide the design of dosing regimens for xenograft models. Serum Aβ40 and Notch target gene modulation in tumours were used to evaluate the degree and duration of target inhibition. Pharmacokinetic and pharmacodynamic correlations with biochemical, immunohistochemical and profiling data were used to demonstrate GSI mechanism of action in xenograft tumours. Key results: Three days of >70% Notch pathway inhibition was sufficient to provide an anti-tumour effect and was well tolerated. GSI-induced conversion of mouse epithelial cells to a secretory lineage was time- and dose-dependent. Anti-tumour efficacy was associated with cell cycle arrest and apoptosis that was in part due to Notch-dependent regulation of mitochondrial homeostasis. Conclusions and implications: Intermittent but potent inhibition of Notch signalling is sufficient for anti-tumour efficacy in these T-ALL models. These findings provide support for the use of GSI in Notch-dependent malignancies and that clinical benefits may be derived from transient but potent inhibition of Notch.

Androgen receptors have been shown to play a critical role in prostate cancer. We used ultrasound imaging techniques to track tumor response to antiandrogen and rapamycin treatment in a prostate-specific Pten-deleted mouse model of cancer. Depletion of androgens by either surgical or chemical castration significantly inhibited tumor growth progression without altering the activation of Akt and mammalian target of rapamycin (mTOR). We also showed for the first time that targeting mTOR along with antiandrogen treatment exhibited additive antitumor effects in vivo when compared with single agents. Our preclinical data suggest that combination of antiandrogens with mTOR inhibitors might be more effective in treating androgen-dependent prostate cancer patients. [Cancer Res 2009;69(18):7466–72]

Mutations in NOTCH1 are frequently detected in patients with T-cell acute lymphoblastic leukemia (T-ALL) and in mouse T-ALL models. Treatment of mouse or human T-ALL cell lines in vitro with γ-secretase inhibitors (GSIs) results in growth arrest and/or apoptosis. These studies suggest GSIs as potential therapeutic agents in the treatment of T-ALL. To determine whether GSIs have antileukemic activity in vivo, we treated near-end-stage Tal1/Ink4a/Arf+/− leukemic mice with vehicle or with a GSI developed by Merck (MRK-003). We found that GSI treatment significantly extended the survival of leukemic mice compared with vehicle-treated mice. Notch1 target gene expression was repressed and increased numbers of apoptotic cells were observed in the GSI-treated mice, demonstrating that Notch1 inhibition in vivo induces apoptosis. T-ALL cell lines also exhibit PI3K/mTOR pathway activation, indicating that rapamycin may also have therapeutic benefit. When GSIs are administered in combination with rapamycin, mTOR kinase activity is ablated and apoptosis induced. Moreover, GSI and rapamycin treatment inhibits human T-ALL growth and extends survival in a mouse xenograft model. This work supports the idea of targeting NOTCH1 in T-ALL and suggests that inhibition of the mTOR and NOTCH1 pathways may have added efficacy.

Inherited genetic risk factors play an important role in cancer. However, other than the Mendelian fashion cancer susceptibility genes found in familial cancer syndromes, little is known about risk modifiers that control individual susceptibility. Here we developed a strategy, parental strain expression mapping, that utilizes the homogeneity of inbred mice and genome-wide mRNA expression analyses to directly identify candidate germ-line modifier genes and pathways underlying phenotypic differences among murine strains exposed to transgenic activation of AKT1 . We identified multiple candidate modifier pathways and, specifically, the glycolysis pathway as a candidate negative modulator of AKT1 -induced proliferation. In keeping with the findings in the murine models, in multiple human prostate expression data set, we found that enrichment of glycolysis pathways in normal tissues was associated with decreased rates of cancer recurrence after prostatectomy. Together, these data suggest that parental strain expression mapping can directly identify germ-line modifier pathways of relevance to human disease.

Prostate cancer remains a major cause of cancer-related mortality. Genetic clues to the molecular pathways driving the most aggressive forms of prostate cancer have been limited. Genetic inactivation of PTEN through either gene deletion or point mutation is reasonably common in metastatic prostate cancer and the resulting activation of phosphoinostide 3-kinase, AKT and mTOR provides a major therapeutic opportunity in this disease as mTOR inhibitors, HSP90 inhibitors and PI3K inhibitors begin to enter clinical development.

Androgen ablation is the primary treatment modality for patients with metastatic prostate cancer; however, the role of androgen receptor signaling in prostate cancer development remains enigmatic. Using a series of genetically defined immortalized and tumorigenic human prostate epithelial cells, we found that introduction of the androgen receptor induced differentiation of transformed prostate epithelial cells to a luminal phenotype reminiscent of organ-confined prostate cancer when placed in the prostate microenvironment. Moreover, androgen receptor expression converted previously androgen-independent, tumorigenic prostate epithelial cells into cells dependent on testosterone for tumor formation. These observations indicate that androgen receptor expression is oncogenic and addictive for the human prostate epithelium.

Understanding the emerging models of adaptive resistance is key to overcoming cancer chemotherapy failure. Using human breast cancer explants, in vitro cell lines, mouse in vivo studies and mathematical modelling, here we show that exposure to a taxane induces phenotypic cell state transition towards a favoured transient ​CD44[superscript Hi]​CD24[superscript Hi] chemotherapy-tolerant state. This state is associated with a clustering of ​CD44 and ​CD24 in membrane lipid rafts, leading to the activation of Src Family Kinase (SFK)/​hemopoietic cell kinase (​Hck) and suppression of apoptosis. The use of pharmacological inhibitors of SFK/​Hck in combination with taxanes in a temporally constrained manner, where the kinase inhibitor is administered post taxane treatment, but not when co-administered, markedly sensitizes the chemotolerant cells to the chemotherapy. This approach of harnessing chemotherapy-induced phenotypic cell state transition for improving antitumour outcome could emerge as a translational strategy for the management of cancer., United States. Dept. of Defense (Breast Cancer Research Program (U.S.) Collaborative Innovator Grant W81XWH-09-1-0700), National Institutes of Health (U.S.) (RO1 1R01CA135242), United States. Dept. of Defense (Breakthrough Award BC132168), American Lung Association (Innovation Award LCD-259932-N), Indo-US Science and Technology Forum (Indo-US Joint Center Grant), American Cancer Society (Postdoctoral Fellowship 122854-PF-12-226-01-CDD)

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.

Background: Telomeres consist of repetitive (TTAGGG) DNA sequences that are maintained by the multisubunit telomerase ribonucleoprotein. Telomerase consists of an RNA, which serves as template for the sequence tracts, and a catalytic subunit that functions in reverse transcription of the RNA template. Cloning and characterization of the human catalytic subunit of telomerase (hTERT) has supported a role in cell transformation. How telomerase activity is regulated, however, is largely unknown. Results: We show here that hTERT associates directly with the c-Abl protein tyrosine kinase. We also found that c-Abl phosphorylates hTERT and inhibits hTERT activity. Moreover, our findings demonstrate that exposure of cells to ionizing radiation induces tyrosine phosphorylation of hTERT by a c-Abl-dependent mechanism. The functional significance of the c-Abl–hTERT interaction is supported by the demonstration that cells deficient in c-Abl show telomere lengthening. Conclusions: The ubiquitously expressed c-Abl tyrosine kinase is activated by DNA double-strand breaks. Our finding of telomere lengthening in c-Abl-deficient cells and the functional interactions between c-Abl and hTERT support a role for c-Abl in the regulation of telomerase function.

The stress-activated protein kinase/c-Jun N-terminal protein kinase (JNK) is induced in response to ionizing radiation and other DNA-damaging agents. Recent studies indicate that activation of JNK is necessary for induction of apoptosis in response to diverse agents. Here we demonstrate that methylmethane sulfonate (MMS)-induced activation of JNK is inhibited by overexpression of the anti-apoptotic protein Bcl-xL, but not by caspase inhibitors CrmA and p35. By contrast, UV-induced JNK activity is insensitive to Bcl-xL. The results demonstrate that treatment with MMS is associated with an increase in tyrosine phosphorylation of related adhesion focal tyrosine kinase (RAFTK)/proline-rich tyrosine kinase 2 (PYK2), an upstream effector of JNK and that this phosphorylation is inhibited by overexpression of Bcl-xL. Furthermore, overexpression of a dominant-negative mutant of RAFTK (RAFTK K-M) inhibits MMS-induced JNK activation. The results indicate that inhibition of RAFTK phosphorylation by MMS in Bcl-xL cells is attributed to an increase in tyrosine phosphatase activity in these cells. Hence, treatment of Bcl-xL cells with sodium vanadate, a tyrosine phosphatase inhibitor, restores MMS-induced activation of RAFTK and JNK. These findings indicate that RAFTK-dependent induction of JNK in response to MMS is sensitive to Bcl-xL, but not to CrmA and p35, by a mechanism that inhibits tyrosine phosphorylation and thereby activation of RAFTK. Taken together, these findings support a novel role for Bcl-xL that is independent of the caspase cascade.

Epidermal growth factor receptor (EGFR), a mediator of mitogenic activity of epidermal growth factor and transforming growth factor-alpha, has been shown to be associated with tumour progression. We have detected a level of EGFR transcript in normal, hyperplastic (BPH) and carcinomatous (CaP) prostate tissues by cytoplasmic dot hybridization. Our results show that the majority of CaP cases (62% of untreated cases and 71% of treated cases) were positive for EGFR mRNA while about 50% of normal and BPH cases were positive for EGFR mRNA. The level of EGFR transcript was significantly higher in the untreated CaP group as compared to the normal group (P < 0.05), while the difference in the normal and BPH groups was not statistically significant.

We have studied the effect of androgenic deprivation on the level of androgen receptor transcript in the rat ventral prostate. The rats were treated with estradiol benzoate, flutamide and [D Trp6, des Gly10]gonadotropin releasing hormone (GnRH) for different time periods. These treatments produced a significant decrease in the weight of prostate. Total RNA isolated from the ventral prostates was hybridized with the cDNA probe for androgen receptor. Densitometric analysis of the autoradiographic signal revealed a rise in the level of androgen receptor RNA following treatment of rats with estradiol benzoate and flutamide. Treatment of rats with [D Trp6, des Gly10] GnRH brought about a transient rise in the level of androgen receptor RNA. Thus, our results indicate that androgenic deprivation up-regulates the level of androgen receptor transcript.

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.

Colorectal cancer (CRC) is one of the leading causes of cancer related mortality. Recent findings from clinical studies showed that immune checkpoint inhibitors are making good progress towards clinical practise. However, existing biomarkers and 3D platforms are not equipped to offer reliable prediction of response of these drugs. We recently engineered a personalized ex vivo systems biology based platform using patient tumors called CANScriptTM (Majumder B et al Nat. Commun. 2015, Goldman A et al Nat. Commun. 2015) and a companion in vivo patient derived immune reconstituted xenograft model (Mi-HTX) preserving multiple critical phenotypic profiles orchestrating tumor-immune cross talk and dysfunction. We further performed baseline characterization of immune markers (CD4, CD8, CD68, CXCR4 and CD45-RO). Intratumor heterogeneity of both M1 and M2 phenotypes were observed irrespective of clinico-pathological features. Baseline CD68 enrichment status was found to be correlated with therapy failure for some patients where clinical outcome (PERCIST) was available. Flow cytometry and microarray analysis showed preservation of diverse sets of functional CD4, CD8 and NK phenotypes along with degranulation markers (CD107a), IFN-γ and FoxP3 in both in vivo and ex vivo tumors reconstituted with ex vivo primed PBMC. The comparative response profiling of these models following treatment with immune checkpoint inhibitors suggested enhanced antitumor effects (as measured by multiple functional parameters like viability, proliferation, morphology and apoptosis). Together, these findings highlight the strengths of immune-competent functional testing tools to stratify patients for precision immunotherapy where direct response prediction biomarkers are still elusive. 1. Majumder B et al. Nat. Commun.6:6169 (2015). 2. Goldman A et al. Nat. Commun. 6:6139 (2015). Citation Format: Pradip K. Majumder, Nilesh Brijwani, Nikita N. Karandikar, Vinod D. Radhakrishna, Muthusamy Oliyarasi, Dency D. Pinto, Babu Balakrishnan, Vasathakumar Sekar, Manoj Rajappa, Debapriya G. Mehrotra, Priyanka Chevur, Priyanka Chevur, Arkasubhra Ghosh, Saravanan Thyiagarajan, Padhma Radhakrishnan, Biswanath Majumder. Delineating immune networks in colorectal cancers to predict effects of immune checkpoint inhibitors using CANScript platform technology preserving tumor immune microenvironment. [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 5129.

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.

The serine/threonine kinase Akt lies at a critical signaling node downstream of phosphatidylinositol-3-kinase and is important in promoting cell survival and inhibiting apoptosis. An Akt inhibitor may be particularly useful for cancers in which increased Akt signaling is associated with reduced sensitivity to cytotoxic agents or receptor tyrosine kinase inhibitors. We evaluated the effect of a novel allosteric Akt inhibitor, MK-2206, in combination with several anticancer agents. In vitro, MK-2206 synergistically inhibited cell proliferation of human cancer cell lines in combination with molecular targeted agents such as erlotinib (an epidermal growth factor receptor inhibitor) or lapatinib (a dual epidermal growth factor receptor/human epidermal growth factor receptor 2 inhibitor). Complementary inhibition of erlotinib-insensitive Akt phosphorylation by MK-2206 was one mechanism of synergism, and a synergistic effect was found even in erlotinib-insensitive cell lines. MK-2206 also showed synergistic responses in combination with cytotoxic agents such as topoisomerase inhibitors (doxorubicin, camptothecin), antimetabolites (gemcitabine, 5-fluorouracil), anti-microtubule agents (docetaxel), and DNA cross-linkers (carboplatin) in lung NCI-H460 or ovarian A2780 tumor cells. The synergy with docetaxel depended on the treatment sequence; a schedule of MK-2206 dosed before docetaxel was not effective. MK-2206 suppressed the Akt phosphorylation that is induced by carboplatin and gemcitabine. In vivo, MK-2206 in combination with these agents exerted significantly more potent tumor inhibitory activities than each agent in the monotherapy setting. These findings suggest that Akt inhibition may augment the efficacy of existing cancer therapeutics; thus, MK-2206 is a promising agent to treat cancer patients who receive these cytotoxic and/or molecular targeted agents. Mol Cancer Ther; 9(7); 1956–67. ©2010 AACR.

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.

Background & Aims The Notch signaling pathway is required for the expansion of undifferentiated pancreatic progenitor cells during embryonic development and has been implicated in the progression of pancreatic ductal adenocarcinoma (PDAC). The interaction of Notch ligands with their receptors promotes a γ-secretase-dependent cleavage of the Notch receptor and release of the Notch intracellular domain, which translocates to the nucleus and activates transcription. We investigated the role of this pathway in PDAC progression. Methods We tested the effects of a γ-secretase inhibitor (GSI) that blocks Notch signaling in PDAC cell lines and a genetically engineered mouse model of PDAC (Kras p53 L/+ mice). Results Notch signaling was activated in PDAC precursors and advanced tumors. The GSI inhibited the growth of premalignant pancreatic duct-derived cells in a Notch-dependent manner. Additionally, in a panel of over 400 human solid tumor-derived cell lines, PDAC cells, as a group, were more sensitive to the GSI than any other tumor type. Finally, the GSI completely inhibited tumor development in the genetically engineered model of invasive PDAC ( P 2 test; compared with mice exposed to vehicle). Conclusions These results suggest that Notch signaling is required for PDAC progression. Pharmacologic targeting of this pathway offers therapeutic potential in this treatment-refractory malignancy.

The Notch pathway is an evolutionarily conserved regulator of cellular differentiation in many tissues during development and later through adult life. It has been reported that particular cancer t...

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