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Table of microinjected drugs

Supplementary Table 1. Hits from the dalotuzumab enhancer screen. Supplementary Table 2. Patient and disease characteristics. Supplementary Table 3. Summary of dose-limiting toxicities. Supplementary Table 4. Summary of clinical efficacy in breast cancer patients.

Microinjection of olaratumab does not inhibit PDGFRα, ERK, or S6 phosphorylation.

All adverse events reported on the study

Supplementary Figure 1. Treatment and pharmacodynamic assessment schedules.

Purpose: Mammalian target of rapamycin (mTOR) inhibition activates compensatory insulin–like growth factor receptor (IGFR) signaling. We evaluated the ridaforolimus (mTOR inhibitor) and dalotuzumab (anti-IGF1R antibody) combination.Experimental Design:In vitro and in vivo models, and a phase I study in which patients with advanced cancer received ridaforolimus (10–40 mg/day every day × 5/week) and dalotuzumab (10 mg/kg/week or 7.5 mg/kg/every other week) were explored.Results: Preclinical studies demonstrated enhanced pathway inhibition with ridaforolimus and dalotuzumab. With 87 patients treated in the phase I study, main dose-limiting toxicities (DLT) of the combination were primarily mTOR-related stomatitis and asthenia at doses of ridaforolimus lower than expected, suggesting blockade of compensatory pathways in normal tissues. Six confirmed partial responses were reported (3 patients with breast cancer); 10 of 23 patients with breast cancer and 6 of 11 patients with ER+/high-proliferative breast cancer showed antitumor activity.Conclusions: Our study provides proof-of-concept that inhibiting the IGF1R compensatory response to mTOR inhibition is feasible with promising clinical activity in heavily pretreated advanced cancer, particularly in ER+/high-proliferative breast cancer (ClinicalTrials.gov identifier: NCT00730379). Clin Cancer Res; 21(1); 49–59. ©2014 AACR.

Supplementary Legend

Distinct drug induced phenotypes and apoptotic responses specific to drug mechanism of action are seen at sites of CIVO microinjection.

Purpose:A persistent issue in cancer drug development is the discordance between robust antitumor drug activity observed in laboratory models and the limited benefit frequently observed when patients are treated with the same agents in clinical trials. Difficulties in accurately modeling the complexities of human tumors may underlie this problem. To address this issue, we developed Comparative In Vivo Oncology (CIVO), which enables in situ investigation of multiple microdosed drugs simultaneously in a patient's tumor. This study was designed to test CIVO's safety and feasibility in patients with soft tissue sarcoma (STS).Patients and Methods:We conducted a single arm, prospective, 13-patient pilot study. Patients scheduled for incisional biopsy or tumor resection were CIVO-injected 1 to 3 days prior to surgery. Saline or microdoses of anticancer agents were percutaneously injected into the tumor in a columnar fashion through each of eight needles. Following excision, drug responses were evaluated in the injected tissue.Results:The primary objective was met, establishing CIVO's feasibility and safety. Device-related adverse events were limited to transient grade 1 nonserious events. In addition, biomarker evaluation of localized tumor response to CIVO microinjected drugs by IHC or with NanoString GeoMx Digital Spatial Profiler demonstrated consistency with known mechanisms of action of each drug, impact on the tumor microenvironment, and historic clinical activity.Conclusions:These results are an advance toward use of CIVO as a translational research tool for early evaluation of investigational agents and drug combinations in a novel approach to phase 0 trials.See related commentary by Sleijfer and Lolkema, p. 3897

Representative images of tumors from patients who received radiation treatment.

Supplementary Figure 3. Change in Ki67 levels with ridaforolimus and dalotuzumab combination therapy

Device performance

Supplementary Figure 2. Combination therapy with ridaforolimus and dalotuzumab potentiates PI3K pathway inhibition and blocks cancer cell proliferation

Clinical Trial Design

Supplementary Figure 4. Responder biomarker hypothesis for ridaforolimus and dalotuzumab?based therapy

Increased phosphorylation of PDGFR� and downstream effectors in an undifferentiated pleomorphic sarcoma displaying a lack of apoptotic response to microdosed doxorubicin.

Purpose: A persistent issue in cancer drug development is the discordance between robust antitumor drug activity observed in laboratory models and the limited benefit frequently observed when patients are treated with the same agents in clinical trials. Difficulties in accurately modeling the complexities of human tumors may underlie this problem. To address this issue, we developed Comparative In Vivo Oncology (CIVO), which enables in situ investigation of multiple microdosed drugs simultaneously in a patient's tumor. This study was designed to test CIVO's safety and feasibility in patients with soft tissue sarcoma (STS). Patients and Methods: We conducted a single arm, prospective, 13-patient pilot study. Patients scheduled for incisional biopsy or tumor resection were CIVO-injected 1 to 3 days prior to surgery. Saline or microdoses of anticancer agents were percutaneously injected into the tumor in a columnar fashion through each of eight needles. Following excision, drug responses were evaluated in the injected tissue. Results: The primary objective was met, establishing CIVO's feasibility and safety. Device-related adverse events were limited to transient grade 1 nonserious events. In addition, biomarker evaluation of localized tumor response to CIVO microinjected drugs by IHC or with NanoString GeoMx Digital Spatial Profiler demonstrated consistency with known mechanisms of action of each drug, impact on the tumor microenvironment, and historic clinical activity. Conclusions: These results are an advance toward use of CIVO as a translational research tool for early evaluation of investigational agents and drug combinations in a novel approach to phase 0 trials. See related commentary by Sleijfer and Lolkema, p. 3897

SUMOylation, the covalent conjugation of small ubiquitin-like modifier (SUMO) proteins to protein substrates, has been reported to suppress type I interferon (IFN1) responses. TAK-981, a selective small molecule inhibitor of SUMOylation, pharmacologically reactivates IFN1 signaling and immune responses against cancers. In vivo treatment of wild type mice with TAK-981 upregulated IFN1 gene expression in blood cells and splenocytes. Ex vivo treatment of mouse and human dendritic cells promoted their IFN1-dependent activation, and vaccination studies in mice demonstrated stimulation of antigen cross-presentation and T cell priming in vivo. TAK-981 also directly stimulated T cell activation, driving enhanced T cell sensitivity and response to antigen ex vivo. Consistent with these observations, TAK-981 inhibited growth of syngeneic A20 and MC38 tumors in mice, dependent upon IFN1 signaling and CD8(+) T cells, and associated with increased intratumoral T and NK cell number and activation. Combination of TAK-981 with anti-PD1 or anti-CTLA4 antibodies improved the survival of mice bearing syngeneic CT26 and MC38 tumors. In conclusion, TAK-981 is a first in class SUMOylation inhibitor that promotes anti-tumor immune responses by activation of IFN1 signaling. TAK-981 is currently being studied in phase I clinical trials (NCT03648372, NCT04074330, NCT04776018 and NCT04381650) for the treatment of patients with solid tumors and lymphomas.

Purpose/Objectives: Tumor responses to cancer treatments are highly context-specific and often involve complex interactions between the anti-cancer therapy, genetically diverse tumor cells, and a heterogeneous tumor microenvironment (TME). All preclinical models fall short in capturing this complexity. CIVO (Comparative In Vivo Oncology) is an intratumoral microdose injection research tool intended to bridge the translational gap between preclinical and clinical studies by enabling in situ assessment of up to 8 oncology drugs or drug combinations simultaneously within a patient’s tumor. The CIVO Phase 0 model was established under FDA’s exploratory IND guidelines for microdosing. A Master Protocol was then developed, enabling ongoing evaluation of multiple investigational drugs and combinations without a need for stand-alone new protocols. Each investigational drug or combination is specified as a substudy of the Master Protocol, thus reducing administrative burden to clinical site staff and creating an infrastructure to ensure quality data and oversight of patient safety. This is a multi-center, open-label Phase 0 Master Protocol designed to study the localized pharmacodynamics (PD) of anti-cancer therapies within the TME when administered intratumorally in microdose quantities via the CIVO device. The safety of intratumoral microdose administration via the CIVO device will also be evaluated. Materials/Methods: Approximately 12 subjects are expected to be enrolled per substudy. All substudies will evaluate subjects ≥18 years with a diagnosis of solid tumors with scheduled surgical intervention. Eligible subjects have at least one lesion (primary or recurrent tumor or effaced metastatic lymph node) ≥2 cm in the shortest diameter that is surface accessible for CIVO injection. Each substudy will define the tumor type and specific eligibility criteria for enrollment. The study visits consist of screening, CIVO injection, surgical intervention, and two follow-up visits. All patients will be injected by the CIVO device containing microdose quantities of drugs specified in respective substudies. The CIVO device can be configured with 3, 5, or 8 needles and the device configuration will be assigned on a per-patient basis, dependent upon lesion size. Following surgical resection, the injected portion of the tumor will undergo central PD biomarker analysis. At the time of submission, the study is open for enrollment with 1 substudy enrolling Head and Neck Squamous Cell Carcinoma (HNSCC) patients and 1 substudy enrolling HNSCC or soft tissue sarcoma patients. The Master Protocol was established to efficiently add substudies and accommodate evaluation of a wider repertoire of new agents in order to continually inform and de-risk drug development via the CIVO platform. Clinical trial information: NCT04541108. Citation Format: Karthik Rajasekaran, Jason G. Newman, Robert G. Maki, Thomas J. Ow, Vikas Mehta, Kenneth R. Gundle, Daniel R. Clayburgh, Ryan J. Li, Mercedes Porosnicu, Cherie-Ann O. Nathan, Alice Tang, Beryl A. Hatton, Kimberly H. Sottero, Gloria Kung, Marc O. Grenley, Kirsten Anderson, Richard A. Klinghoffer. A phase 0 master protocol utilizing a novel intratumoral microdosing approach for simultaneously evaluating multiple drugs and drug combinations in patients with solid tumors [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 CT216.

Purpose/Objectives: Individual tumor complexity and the emergence of treatment resistance mechanisms have proven challenging for oncology drug development, and methods for prioritizing combination therapy approaches are needed. The CIVO (Comparative In Vivo Oncology) platform can simultaneously deliver up to 8 drugs and combinations in microdose amounts to distinct, trackable regions within a single intact tumor, enabling studies that can assess multiple drugs and combinations directly in patients during early drug development. This study employed CIVO to assess two combination strategies incorporating the STING (Stimulator of Interferon Genes) agonist TAK-676 and chemotherapies capable of inducing immunogenic cell death. We evaluated TAK-676’s ability to elicit pharmacodynamic (PD) changes suggestive of anti-tumor immune activation within the tumor microenvironment (TME) in a syngeneic mouse model, both as monotherapy and in combination with cisplatin and 5-FU or cisplatin and paclitaxel chemotherapy doublets. Materials/Methods: TAK-676 and chemotherapy combinations were simultaneously microdosed via CIVO in a syngeneic mouse melanoma model (YUMM1.7). Successful injections were visualized after administration, during sample processing, and in biomarker-stained tissue sections via a fluorescent tracking marker co-injected through each needle, and PD responses were assessed via immunohistochemistry and in situ hybridization following tumor resection. Results: Drug mechanism of action-specific biomarker activity was evident as early as 4 hours after injection. TAK-676 induced robust elevation of pIRF3 and CXCL10 along with IFNγ from both T and NK cell compartments, indicating STING pathway activation. Phospho-histone H3 accumulation was noted in response to paclitaxel-induced mitotic arrest. 5-FU and cisplatin induced localized DNA damage, visible via elevated phospho-histone H2AX-positive nuclei. Cytotoxic T lymphocyte (CTL) accumulation was detected around TAK-676 injection sites, likely recruited from the local TME via induction of chemokines such as CXCL10. Early tumor cell apoptosis and induction of pro-inflammatory cytokines such as CXCL10 were detected in response to both triplet combinations. CTL activation was enhanced in response to both triplet combinations by 24 hours, whereas CTL enrichment at levels greater than induced by TAK-676 alone was noted specifically in response to the triplet combination with cisplatin and paclitaxel by 72 hours. Conclusion: These studies highlight TAK-676’s potential to promote anti-tumor immunity and the utility of the CIVO platform to reveal and characterize combination-specific responses. This application of CIVO is being further evaluated in an ongoing Phase 0 trial in patients with head and neck squamous cell carcinoma (NCT04541108). Citation Format: Beryl A. Hatton, Marc Grenley, Sally Ditzler, Richard A. Klinghoffer, Allison J. Berger, Karim S. Malek, Richard C. Gregory, Neil B. Lineberry. Intratumoral microdosing via the CIVO® Platform reveals anti-tumor immune responses induced by the STING Agonist TAK-676 alone and in combination with chemotherapies [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 620.

Purpose/Objectives: The human tumor microenvironment (TME) has a dramatic impact on cancer prognosis and therapeutic response, but accurate models of the native TME do not exist. The Comparative In Vivo Oncology (CIVO) platform was developed as a means to assess the effect of investigational agents on the native TME in a Phase 0 microdosing study. CIVO was clinically validated using approved agents and is used for the first time here to assess the impact of an investigational agent - the SUMOylation inhibitor TAK-981 - on the native human TME in HNSCC. Materials/Methods: Eligible subjects have a confirmed HNSCC diagnosis, ECOG 0-2, and planned surgical resection. Injectable tumors were at the primary site or within cervical lymph nodes but had to be surface-accessible and ≥ 2cm. TAK-981 or control microdoses were simultaneously administered via a CIVO device and co-injected with a fluorescent tracking marker for injection site identification and visualization. Tumors were resected 24 or 72 hours after injection, processed, and then analyzed at a central site. Multiplexed biomarker staining and molecular profiling via GeoMx Digital Spatial Profiling were performed to capture pharmacodynamic responses in the native TME. Results: As of January 2022, 8 subjects provided informed consent and were enrolled, and no adverse events associated with the injection procedure or microdoses have been reported. Biomarker analysis demonstrated TAK-981 distribution around the injection site accompanied by reduction of SUMOylation. Dose-dependent elevation of IFN1 signaling was also observed in TAK-981-exposed areas within the TME. Elevated IFN1 signaling was accompanied by TME reconfiguration, with increased macrophage M1 polarization and activation of dendritic cells, NK cells, and CD8+ T cells. TAK-981 exposure was also associated with upregulation of CXCL10, PD-L1, and an IFNγ gene expression signature predictive of response to immune checkpoint blockade. Conclusion: IT microdosing with CIVO provided early insights into complex functional responses induced by the investigational agent TAK-981 that can only be accurately evaluated in the intact, native TME of a patient’s tumor. SUMO pathway inhibition in HNSCC tumors following TAK-981 exposure led to functional activation of multiple immune cell types, effectively shifting the local TME toward an inflamed “hot” state, highlighting TAK-981’s potential as an immune stimulating agent for treating patients with solid tumors. These data were generated while TAK-981 was still in Phase I dose escalation trials (via IV administration), highlighting CIVO’s ability to safely study investigational agents. Further evaluation of TAK-981 alone and in combination with other agents is ongoing in this Phase 0 CIVO microdosing trial. Citation Format: Jeffrey Houlton, Harrison Cash, Haodong Xu, Paul L. Swiecicki, Keith Casper, Steven B. Chinn, Daniel R. Clayburgh, Ryan J. Li, Robert J. Christian, Aaron Halfpenny, Annemieke van Zante, Beryl A. Hatton, Kimberly Sottero, Marc O. Grenley, Connor Burns, Jason Frazier, Jonathan Derry, Gloria Kung, Emily Beirne, Nathan J. Schauer, Atticus Turner, Wendy Jenkins, Kirsten Anderson, Richard A. Klinghoffer, Dennis Huszar, Allison Berger, Karuppiah Kannan. Intratumoral (IT) microdosing of the investigational SUMOylation Inhibitor TAK-981 in a phase 0 CIVO trial demonstrates the reactivation of type I Interferon (IFN1) signaling in head and neck squamous cell carcinoma (HNSCC) [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 CT139.

A persistent issue in cancer drug development is the discordance between robust antitumor drug activity observed in laboratory models and the limited benefit frequently observed when patients are treated with the same agents in clinical trials. Difficulties in accurately modeling the complexities of human tumors may underlie this problem. To address this issue, we developed ComparativeWe conducted a single arm, prospective, 13-patient pilot study. Patients scheduled for incisional biopsy or tumor resection were CIVO-injected 1 to 3 days prior to surgery. Saline or microdoses of anticancer agents were percutaneously injected into the tumor in a columnar fashion through each of eight needles. Following excision, drug responses were evaluated in the injected tissue.The primary objective was met, establishing CIVO's feasibility and safety. Device-related adverse events were limited to transient grade 1 nonserious events. In addition, biomarker evaluation of localized tumor response to CIVO microinjected drugs by IHC or with NanoString GeoMx Digital Spatial Profiler demonstrated consistency with known mechanisms of action of each drug, impact on the tumor microenvironment, and historic clinical activity.These results are an advance toward use of CIVO as a translational research tool for early evaluation of investigational agents and drug combinations in a novel approach to phase 0 trials.

A new generation of both systemic and targeted CD137 agonists are entering clinical development following agonistic anti-CD137 (4-1BB) antibody development nearly a decade ago. However, each of these molecules rely on biologic agents with potentially suboptimal properties for CD137 agonism due to their relatively large size and long circulating half-lives which may limit their tissue penetration, cause sustained agonism resulting in overstimulation and activation-induced lymphocyte cell death. Fully synthetic constrained bicyclic peptides (Bicycles™) with antibody-like affinities and target selectivity are uniquely suited to circumvent limitations of other targeted CD137 agonistic therapeutics. BT7480 and BCY11864 are tumor targeted immune cell agonists (TICA) designed to deliver a highly potent CD137 agonist to Nectin-4 overexpressing tumors with a flexible dosing schedule maximizing anti-tumor activity while circumventing the need for continuous systemic exposure. The Comparative In Vivo Oncology (CIVO) platform has been developed to enable in situ investigation of multiple microdosed drugs simultaneously in human tumors with safety and feasibility of this platform recently demonstrated in patients with soft tissue sarcomas. CIVO percutaneously injects drug microdoses directly into tumor tissue as trackable columns where tissue can be analyzed after resection for the effect of the drug treatment in the tumor. Here we report on an evaluation of the feasibility of using the CIVO -platform to demonstrate the mechanism of action of our tumor target-dependent CD137 agonist TICAs. Both BT7480 and BCY11864 demonstrate extremely potent Nectin-4-dependent CD137 agonism in primary human PBMC/tumor cell co-culture assays. Systemic administration of BT7480 in Nectin-4 expressing tumors in immunocompetent mice leads to profound reprogramming of the tumor immune microenvironment including a rapid increase in T cell chemotactic cytokine transcripts and macrophage gene signatures followed by an increase in cytotoxic cell gene signature and increase in intratumoral CD8+ T cells. CIVO was used to demonstrate the in vivo mechanism of action of a Nectin-4/CD137 TICA. Micro-injection of BCY11864 into Nectin-4 expressing tumors in immunocompetent mice led to dose dependent induction of immune activation markers in the tumor microenvironment and activation of the tumor resident cytotoxic T cells was evident 24 hours after BCY11864 micro-injection. Akin to our findings with transcriptional profiling of the tumor response to BT7480, BCY11864 activity was shown not to be limited to T cells but also included other CD137 positive cell populations such as myeloid and NK cells. We hypothesize that tumor targeted CD137 agonism may lead to initial myeloid cell response that enhances the cytotoxic T cell recruitment and activation in the tumor tissue. Citation Format: Johanna Lahdenranta, Stephen J. Blakemore, Kristen Hurov, Marc O. Grenley, Punit Upadhyaya, Carly Campbell, Elizabeth Repash, Eric Haines, Philip E. Brandish, Richard A. Klinghoffer, Nicholas Keen. Microinjection of Nectin-4/CD137 tumor-targeted immune cell agonist (TICA™) activates the local tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1724.

Aberrant regulation of BCL-2 family members enables evasion of apoptosis and tumor resistance to chemotherapy. BCL-2 and functionally redundant counterpart, MCL-1, are frequently over-expressed in high-risk diffuse large B-cell lymphoma (DLBCL). While clinical inhibition of BCL-2 has been achieved with the BH3 mimetic venetoclax, anti-tumor efficacy is limited by compensatory induction of MCL-1. Voruciclib, an orally bioavailable clinical stage CDK-selective inhibitor, potently blocks CDK9, the transcriptional regulator of MCL-1. Here, we demonstrate that voruciclib represses MCL-1 protein expression in preclinical models of DLBCL. When combined with venetoclax in vivo, voruciclib leads to model-dependent tumor cell apoptosis and tumor growth inhibition. Strongest responses were observed in two models representing high-risk activated B-cell (ABC) DLBCL, while no response was observed in a third ABC model, and intermediate responses were observed in two models of germinal center B-cell like (GCB) DLBCL. Given the range of responses, we show that CIVO, a multiplexed tumor micro-dosing technology, represents a viable functional precision medicine approach for differentiating responders from non-responders to BCL-2/MCL-1 targeted therapy. These findings suggest that the combination of voruciclib and venetoclax holds promise as a novel, exclusively oral combination therapy for a subset of high-risk DLBCL patients.

Spontaneously occurring soft tissue sarcoma (STS) is relatively common in canine cancer patients. Because of the similarities to human disease, canine STSs are a valuable and readily available resource for the study of new therapeutics. In this study, a canine patient-derived xenograft (PDX) model, CDX-STS2, was established. The CDX-STS2 model was engrafted and expanded for systemic administration studies with chemotherapeutic agents commonly used to treat STS, including doxorubicin, docetaxel and gemcitabine. Immunohistochemistry for drug-specific biomarkers and tumour growth measurement revealed tumour sensitivity to doxorubicin and docetaxel, whereas gemcitabine had no effect on tumour growth. Although many human PDX tumour models have been established, relatively few canine PDX models have been reported to date. CDX-STS2 represents a new STS PDX research model of canine origin that will be useful in bridging preclinical research with clinical studies of STS in pet dogs.

Purpose To facilitate decision making in the oncology clinic, technologies have recently been developed to independently inject and assess multiple anticancer agents directly in a patient's tumor. To increase the flexibility of this approach beyond histological readouts of response, contrast-enhanced MRI was evaluated for the detection of cell death in living tumors after injection. Methods A six-needle arrayed microinjection device designed to provide head-to-head comparisons of chemotherapy responses in living tumors was used. Xenografted non-Hodgkin lymphoma tumors in athymic Nude-Foxn1nu mice were injected either with different doses of vincristine or with one needle each of vincristine, doxorubicin, bendamustine, prednisolone, mafosfamide, and a vehicle control. To assess drug responses, measurements of enhancement by T1-weighted contrast-enhanced MRI were made for individual sites at 24, 48, and 72 h after injection. For comparison, histological evaluations of cell death were obtained after tumor resection. Results Measurements of MRI enhancement at injection sites showed a significant (P

The vision of a precision medicine–guided approach to novel cancer drug development is challenged by high intratumor heterogeneity and interpatient diversity. This complexity is rarely modeled accurately during preclinical drug development, hampering predictions of clinical drug efficacy. To address this issue, we developed Comparative In Vivo Oncology (CIVO) arrayed microinjection technology to test tumor responsiveness to simultaneous microdoses of multiple drugs directly in a patient's tumor. Here, in a study of 18 canine patients with soft tissue sarcoma (STS), CIVO captured complex, patient-specific tumor responses encompassing both cancer cells and multiple immune infiltrates following localized exposure to different chemotherapy agents. CIVO also classified patient-specific tumor resistance to the most effective agent, doxorubicin, and further enabled assessment of a preclinical autophagy inhibitor, PS-1001, to reverse doxorubicin resistance. In a CIVO-identified subset of doxorubicin-resistant tumors, PS-1001 resulted in enhanced antitumor activity, increased infiltration of macrophages, and skewed this infiltrate toward M1 polarization. The ability to evaluate and cross-compare multiple drugs and drug combinations simultaneously in living tumors and across a diverse immunocompetent patient population may provide a foundation from which to make informed drug development decisions. This method also represents a viable functional approach to complement current precision oncology strategies. Cancer Res; 77(11); 2869–80. ©2017 AACR.

The complex interplay between a drug, the tumor and the surrounding microenvironment is a critical determinant in how a patient will respond to their selected cancer therapy. Yet to date, this complex response has been difficult to evaluate prior to late stage clinical investigation. To enable a means for testing investigational agents earlier in the development process but still directly in human patients, in a way that limits the risk of adverse effects and provides an indication of efficacy, Presage Biosciences developed a technology called CIVO. This platform allows for simultaneous assessment of multiple drugs or drug combinations directly in a single solid tumor, in the context where the drugs are ultimately intended to be used, directly in the patient. Here we demonstrate the potential for using the CIVO platform in Phase 0 microdosing studies to detect complex responses to investigational agents. In this study, we used the CIVO platform to assess the impact of TAK-981 on the native tumor immune microenvironment of animal models. TAK-981 is a novel and selective small molecule inhibitor of the SUMOylation enzymatic cascade currently in Phase I clinical trials. SUMOylation is a reversible post-translational modification that regulates protein function by covalent attachment of a small ubiquitin-like modifier (SUMO) protein to protein substrates. TAK-981 was microinjected into tumors from a syngeneic mouse model of B cell lymphoma and responses assessed via immunohistochemistry and in situ hybridization following tumor resection. An early inflammatory response was evident by 24 hours, including the accumulation of neutrophils, inflammatory macrophages and a Type I interferon response. The chemokine IP10 was secreted around TAK-981 injection sites and was accompanied by the accumulation of cytotoxic T lymphocytes, likely recruited from the local tumor microenvironment. A localized cell death response was observed proximal to TAK-981 injection sites by 72 hours and was likely induced by the granzyme B-bearing cytotoxic T cells enriched at TAK-981 sites. Abscopal studies demonstrated that the local immune modulation induced by TAK-981 translated into an adaptive immune response. The results from this study were consistent with findings from systemically dosed in vivomouse efficacy studies carried out at Takeda, demonstrating that the local responses to agents microdosed intratumorally via CIVO are predictive of responses induced by systemic drug exposure. These studies highlight the unique capability of TAK-981 to promote antitumor immunity, which may be further evaluated using the CIVO platform in a Phase 0 trial in human solid tumor patients. Citation Format: Beryl A. Hatton, Marc Grenley, James Garnsey, Vaishali Shinde, Dennis Huszar, Connor Burns, Sally Ditzler, Angela Merrell, Joyoti Dey, Emily Beirne, Richard A. Klinghoffer. Direct intratumoral microdosing via the CIVO® platform reveals anti-tumor immune responses induced by the SUMO inhibitor TAK-981 [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 4136.

Background: CIVO microdosing studies performed in patient tumors in situ allow drug developers to assess localized tumor and microenvironment responses to multiple agents without having to expose patients to high systemic drug levels. By concentrating microdoses of multiple different drugs within a living tumor in situ, it is possible to compare tumor and immune responses in spatially resolved regions of the same tumor. This type of early phase (Phase 0) clinical study represents a new path for drug developers to gain insight into drug efficacy, tumor associated immune cell modulation, biomarker discovery and validation, and microenvironment interactions for new drugs earlier in the drug development process. Traditionally, analysis of FFPE samples from these microdosing studies involve routine immunohistochemistry, immunofluorescence, and in situ assays that reveal changes in protein and RNA expression. These assays offer tissue wide protein and gene expression information but have limited multiplexing capabilities and dynamic range, as well as rapidly consume precious trial samples. Novel technologies such as NanoString’s GeoMxTM Digital Spatial Profiler (DSP) enable high-plex spatially resolved analysis of proteins and RNA transcripts in single FFPE tissue sections. In this proof of principle study, we utilized GeoMxTM Digital Spatial Profiler for protein and RNA expression on single FFPE sections from patient sarcoma tumors that were microdosed with multiple FDA approved drugs. Methods: Single FFPE sections from microdosed patient tumor samples were IF stained for DNA and CD3 and whole slide imaged. 600-micron and 100-micron diameter regions of interests (ROIs) were selected for DSP analysis within drug and control microinjection sites. Imaging and barcode counts were performed using GeoMxTM DSP and nCounter systems. Results: DSP protein analysis highlighted phospho-S6 and phospho-ERK upregulation in response to doxorubicin compared to vehicle site. Additionally, ROIs sampled along the doxorubicin exposure gradient showed a dose-dependent reduction of phosphorylation of both S6 and ERK proteins. DSP RNA analysis revealed drug specific transcript regulation of multiple genes in microdosed tumors, including upregulation of chemokines CXCL9 and CXCL10 at sites of doxorubicin and aldesleukin injection but not to other chemotherapy agents. Conclusions: Early phase CIVO microdosing studies combined with high-plex DSP opens the door to generating multi-omics data for multiple microdosed drugs within small patient studies. Analysis of protein and RNA expression using DSP enabled collection of targeted region of interest high density data from single FFPE sections, conserving precious patient biopsy samples. Through continued expansion of the GeoMxTM DSP analyte panels, collecting an ever-increasing depth of protein and gene expression data is possible in Phase 0 CIVO microdosing studies. Citation Format: Gary B. Deutsch, Seth M. Pollack, Matthew J. Thompson, Kenneth R. Gundle, Jessica A. Bertout, Jason P. Frazier, Emily Beirne, Marc O. Grenley, JingJing Gong, Yan Liang, Joseph M. Beechem, Richard A. Klinghoffer, Robert G. Maki. High-plex spatial profiling analysis of multidrug CIVO microdose studies in cancer patients [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 2155.

While advances in high-throughput screening have resulted in increased ability to identify synergistic anti-cancer drug combinations, validation of drug synergy in the in vivo setting and prioritization of combinations for clinical development remain low-throughput and resource intensive. Furthermore, there is currently no viable method for prospectively assessing drug synergy directly in human patients in order to potentially tailor therapies. To address these issues we have employed the previously described CIVO platform and developed a quantitative approach for investigating multiple combination hypotheses simultaneously in single living tumors. This platform provides a rapid, quantitative and cost effective approach to compare and prioritize drug combinations based on evidence of synergistic tumor cell killing in the live tumor context. Using a gemcitabine resistant model of pancreatic cancer, we efficiently investigated nine rationally selected Abraxane-based combinations employing only 19 xenografted mice. Among the drugs tested, the BCL2/BCLxL inhibitor ABT-263 was identified as the one agent that synergized with Abraxane® to enhance acute induction of localized apoptosis in this model of human pancreatic cancer. Importantly, results obtained with CIVO accurately predicted the outcome of systemic dosing studies in the same model where superior tumor regression induced by the Abraxane/ABT-263 combination was observed compared to that induced by either single agent. This supports expanded use of CIVO as an in vivo platform for expedited in vivo drug combination validation and sets the stage for performing toxicity-sparing drug combination studies directly in cancer patients with solid malignancies.

SummaryMolecular-level understanding of body weight control is essential for combating obesity. We show that female mice lacking tyrosine phosphatase epsilon (RPTPe) are protected from weight gain induced by high-fat food, ovariectomy, or old age and exhibit increased whole-body energy expenditure and decreased adiposity. RPTPe-deficient mice, in particular males, exhibit improved glucose homeostasis. Female nonobese RPTPe-deficient mice are leptin hypersensitive and exhibit reduced circulating leptin concentrations, suggesting that RPTPe inhibits hypothalamic leptin signaling in vivo. Leptin hypersensitivity persists in aged, ovariectomized, and high-fat-fed RPTPe-deficient mice, indicating that RPTPe helps establish obesity-associated leptin resistance. RPTPe associates with and dephosphorylates JAK2, thereby downregulating leptin receptor signaling. Leptin stimulation induces phosphorylation of hypothalamic RPTPe at its C-terminal Y695, which drives RPTPe to downregulate JAK2. RPTPe is therefore an inhibitor of hypothalamic leptin signaling in vivo, and provides controlled negative-feedback regulation of this pathway following its activation.

The vision of a precision medicine-guided approach to novel cancer drug development is challenged by high intra-tumor heterogeneity, interpatient diversity, and complex interactions between tumor cells and the surrounding microenvironment. Attempts to model this complexity through the use of ex vivo systems have failed to accurately represent tumor biology, and resulting predictions of clinical drug efficacy have met with limited success. The era of genomics, highlighted by remarkable advances in high-throughput next generation sequencing technology, has sparked hope that this issue can be solved through a precision medicine approach to cancer treatment: matching the right drugs to the right patients. However, despite a few noteworthy successes pairing targeted agents with oncogenic driver mutations, genome-based precision medicine has been limited by an incomplete understanding of the relationship between cancer mutations and drug responsiveness. To overcome these issues, we developed CIVO (Comparative In Vivo Oncology), an arrayed microinjection technology that evaluates tumor responsiveness to microdoses of multiple drugs simultaneously and directly in a patient's tumor. CIVO consists of a multidrug injection device, fluorescent tracking microspheres which accurately denote the position of each drug injection site, and automated image analysis tools capable of quantifying both tumor cell and microenvironmental biomarkers of response to local drug exposure. Here we present preliminary data on the first 4 patients from our clinical feasibility study in soft tissue sarcoma. No unexpected adverse events related to microdose injection were observed. Consistent with historical clinical data, front line agent doxorubicin induced localized increases in markers for DNA damage, tumor cell apoptosis, and macrophage infiltration, whereas gemcitabine did not induce any observable local responses. Doxorubicin also induced tumor cell responses that suggest potential mechanisms of resistance to single agent therapy, including upregulation of PDGFαR and ERK phosphorylation. Multidrug investigation via intratumoral microinjection with CIVO in patients with soft tissue sarcoma appears safe and feasible. The ability to evaluate and cross-compare multiple drugs and drug combinations simultaneously in living tumors across a diverse immune competent patient population provides an innovative and exciting functional approach to complement current precision oncology strategies and enables deep characterization of cancer biology in early drug development. Citation Format: Matthew J. Thompson, Darin J. Davidson, Jessica A. Bertout, Kimberly H. Sottero, Marc O. Grenley, Emily Beirne, Richard A. Klinghoffer, Seth M. Pollack. Multidrug analyses in cancer patients via intratumoral microdosing with CIVO: A functional approach to precision oncology [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 CT142.

e23569Background: The vision of a precision medicine-guided approach to novel cancer drug development is challenged by high intra-tumor heterogeneity, interpatient diversity, and complex interactio...

Along with silencing intended target genes, transfected siRNAs regulate numerous unintended transcripts through a mechanism in which the equivalent of a microRNA-like seed region in the siRNA recognizes complementary sequences in transcript 3′ UTRs. Amelioration of this off-target silencing would lead to more accurate interpretation of RNA interference (RNAi) experiments and thus greatly enhance their value. We tested whether lentivirus-mediated delivery of shRNA is prone to the sequence-based off-target activity prevalent in siRNA experiments. We compared target gene silencing and overall impact on global gene expression caused by multiple sequences delivered as both transfected siRNAs and lentivirus vector-expressed shRNAs. At equivalent levels of target gene silencing, signatures induced by shRNAs were significantly smaller than those induced by cognate siRNAs and arose less frequently from seed region activity. Interestingly, the low level of seed region-based off-target activity exhibited by shRNAs resulted in down-regulation of transcripts that were largely distinct from those regulated by siRNAs. On the basis of these observations, we recommend lentivirus-mediated RNAi for pathway profiling experiments that measure whole genome transcriptional readouts as well as for large-scale screens when resources for extensive follow up are limited.

To facilitate decision making in the oncology clinic, technologies have recently been developed to independently inject and assess multiple anticancer agents directly in a patient's tumor. To increase the flexibility of this approach beyond histological readouts of response, contrast-enhanced MRI was evaluated for the detection of cell death in living tumors after injection.A six-needle arrayed microinjection device designed to provide head-to-head comparisons of chemotherapy responses in living tumors was used. Xenografted non-Hodgkin lymphoma tumors in athymic Nude-Foxn1(nu) mice were injected either with different doses of vincristine or with one needle each of vincristine, doxorubicin, bendamustine, prednisolone, mafosfamide, and a vehicle control. To assess drug responses, measurements of enhancement by T1-weighted contrast-enhanced MRI were made for individual sites at 24, 48, and 72 h after injection. For comparison, histological evaluations of cell death were obtained after tumor resection.Measurements of MRI enhancement at injection sites showed a significant (P 0.001) positive regression slope as a function of vincristine dose. Average MRI measurements were closely correlated with cell death by hematoxylin and eosin staining (R = 0.81; P = 0.001).Contrast-enhanced MRI has the potential to replace or augment histological analyses of tumor responses to microinjected doses of chemotherapy agents with potential application in selecting optimal chemotherapy regimens. Magn Reson Med 76:946-952, 2016. © 2015 Wiley Periodicals, Inc.

A fundamental problem in cancer drug development is that antitumor efficacy in preclinical cancer models does not translate faithfully to patient outcomes. Much of early cancer drug discovery is performed under in vitro conditions in cell-based models that poorly represent actual malignancies. To address this inconsistency, we have developed a technology platform called CIVO, which enables simultaneous assessment of up to eight drugs or drug combinations within a single solid tumor in vivo. The platform is currently designed for use in animal models of cancer and patients with superficial tumors but can be modified for investigation of deeper-seated malignancies. In xenograft lymphoma models, CIVO microinjection of well-characterized anticancer agents (vincristine, doxorubicin, mafosfamide, and prednisolone) induced spatially defined cellular changes around sites of drug exposure, specific to the known mechanisms of action of each drug. The observed localized responses predicted responses to systemically delivered drugs in animals. In pair-matched lymphoma models, CIVO correctly demonstrated tumor resistance to doxorubicin and vincristine and an unexpected enhanced sensitivity to mafosfamide in multidrug-resistant lymphomas compared with chemotherapy-naïve lymphomas. A CIVO-enabled in vivo screen of 97 approved oncology agents revealed a novel mTOR (mammalian target of rapamycin) pathway inhibitor that exhibits significantly increased tumor-killing activity in the drug-resistant setting compared with chemotherapy-naïve tumors. Finally, feasibility studies to assess the use of CIVO in human and canine patients demonstrated that microinjection of drugs is toxicity-sparing while inducing robust, easily tracked, drug-specific responses in autochthonous tumors, setting the stage for further application of this technology in clinical trials.

Receptor tyrosine kinases (RTKs) direct diverse cellular and developmental responses by stimulating a relatively small number of overlapping signaling pathways. Specificity may be determined by RTK expression patterns or by differential activation of individual signaling pathways. To address this issue we generated knock-in mice in which the extracellular domain of the mouse platelet-derived growth factor alpha receptor (PDGFalphaR) is fused to the cytosolic domain of Drosophila Torso (alpha(Tor)) or the mouse fibroblast growth factor receptor 1 (alpha(FR)). alpha(Tor) homozygous embryos exhibit significant rescue of neural crest and angiogenesis defects normally found in PDGFalphaR-null embryos yet fail to rescue skeletal or extraembryonic defects. This phenotype was associated with the ability of alpha(Tor) to stimulate the mitogen-activated protein (MAP) kinase pathway to near wild-type levels but failure to completely activate other pathways, such as phosphatidylinositol (PI) 3-kinase. The alpha(FR) chimeric receptor fails to rescue any aspect of the PDGFalphaR-null phenotype. Instead, alpha(FR) expression leads to a gain-of-function phenotype highlighted by ectopic bone development. The alpha(FR) phenotype was associated with a failure to limit MAP kinase signaling and to engage significant PI3-kinase response. These results suggest that precise regulation of divergent downstream signaling pathways is critical for specification of RTK function.

Targeted therapies designed to inhibit hyperactive oncogenic signaling have demonstrated some encouraging clinical responses. However, most of the time these responses are not durable as tumors “work around” pathway inhibition, often reactivating the inhibited pathway, leading to drug resistance and disease relapse. New technologies that enable more efficient discovery and development of “suites” of drugs that comprehensively inhibit multiple pathway nodes are needed. The SABRE (Splice Acceptor Brilliant Reporter) platform was developed to provide an unbiased method to screen large compound libraries and dramatically improve the speed and efficiency of which novel targeted therapeutics can be identified. SABRE is built off the premise that oncogenic output is exerted through changes in gene transcription and that such changes can be harnessed as powerful reporters of pathway activation status. SABRE utilizes the power of gene trap technology coupled with a drug selection process to isolate cells that generate a robust “off to on” signal in response to target or pathway inhibition. Via massively parallel comparative analysis, multiple traps let nature provide the best reporter for further analysis and drug discovery. In this proof-of-concept study, we employed the SABRE technology to identify insertion sites that are specifically regulated by the MAPK pathway. Experiments were performed using the human BRAFV600E mutant melanoma cell line A375. SABRE lentiviral transduced A375 cells were treated with trametinib, a MEK inhibitor, and clones were isolated that emitted a positive luciferase signal upon drug treatment. To determine if these reporters were MAPK pathway specific, the platform was miniaturized to a 1536 well format and used to screen a 6000+ compound library. Results from the screen found that 70% (28/40) of the top drug hits were known to directly modulate the MAPK pathway. Since drug resistance is a common occurrence in melanoma, we generated a “resistant” A375 SABRE reporter line to increasing concentrations of vemurafenib and interrogated the top drug candidates identified in the screen. As expected, the resistant SABRE line failed to respond to BRAFV600E specific inhibitors but continued to respond to downstream MEK inhibitors, which is helping to delineate the mechanism of resistance. We were also interested in the screening hits that were not previously known to influence the MAPK pathway including bafetinib and a Tie2 inhibitor. In follow-up testing, we found that these drugs induced robust and titratable reporter signals in both the SABRE A375 vemurafenib sensitive and resistant lines. In addition, treatment of either bafetinib or the Tie2 inhibitor reduced ERK phosphorylation in the vemurafenib resistant cells, suggesting that these drugs could be useful in the treatment of drug resistant melanomas. Studies are currently underway to determine the in vivo efficacy of these drugs in A375 SABRE reporter xenograft models. Citation Format: Shelli M. Morris, Andrew J. Mhyre, Savanna S. Carmack, Connor Burns, Marc Ferrer, Wenjuan Ye, James M. Olson, Richard A. Klinghoffer. The SABRE platform: A novel, unbiased technology for drug discovery and prioritization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4023. doi:10.1158/1538-7445.AM2017-4023

Purpose: Mammalian target of rapamycin (mTOR) inhibition activates compensatory insulin–like growth factor receptor (IGFR) signaling. We evaluated the ridaforolimus (mTOR inhibitor) and dalotuzumab (anti-IGF1R antibody) combination. Experimental Design: In vitro and in vivo models, and a phase I study in which patients with advanced cancer received ridaforolimus (10–40 mg/day every day × 5/week) and dalotuzumab (10 mg/kg/week or 7.5 mg/kg/every other week) were explored. Results: Preclinical studies demonstrated enhanced pathway inhibition with ridaforolimus and dalotuzumab. With 87 patients treated in the phase I study, main dose-limiting toxicities (DLT) of the combination were primarily mTOR-related stomatitis and asthenia at doses of ridaforolimus lower than expected, suggesting blockade of compensatory pathways in normal tissues. Six confirmed partial responses were reported (3 patients with breast cancer); 10 of 23 patients with breast cancer and 6 of 11 patients with ER+/high-proliferative breast cancer showed antitumor activity. Conclusions: Our study provides proof-of-concept that inhibiting the IGF1R compensatory response to mTOR inhibition is feasible with promising clinical activity in heavily pretreated advanced cancer, particularly in ER+/high-proliferative breast cancer (ClinicalTrials.gov identifier: NCT00730379). Clin Cancer Res; 21(1); 49–59. ©2014 AACR.

We tested the hypothesis that Src family kinases (SFK) contribute to c-Cbl-mediated degradation of the platelet-derived growth factor (PDGF) alpha receptor (alphaPDGFR). Using either a receptor mutant that does not engage SFKs (F72/74), or cells that that lack SFKs, we found that SFKs contributed to degradation of the alphaPDGFR. Overexpression of c-Cbl also reduced the receptor half-life, but only if the receptor was able to engage SFKs. In cultured cells, prolonging the half-life of the receptor correlated with enhanced signaling and more efficient S phase entry, whereas accelerating receptor degradation had the opposite effect. Consistent with these tissue culture findings, there was a statistically significant increase in the onset of a proliferative retinal disease when animals were injected with cells expressing the F72/74 receptor, as compared with cells expressing the WT receptor. Our findings suggest that SFKs cooperate with c-Cbl to negatively regulate the alphaPDGFR, and that the SFK/c-Cbl suppression of alphaPDGFR output is relevant to the onset and progression of a proliferative disease.

Eph receptor tyrosine kinases and their corresponding surface-bound ligands, the ephrins, provide cues to the migration of cells and growth cones during embryonic development. Here we show that ephrin-A5, which is attached to the outer leaflet of the plasma membrane by a glycosyl-phosphatidylinositol-anchor, induces compartmentalized signaling within a caveolae-like membrane microdomain when bound to the extracellular domain of its cognate Eph receptor. The physiological response induced by this signaling event is concomitant with a change in the cellular architecture and adhesion of the ephrin-A5-expressing cells and requires the activity of the Fyn protein tyrosine kinase. This study stresses the relevance of bidirectional signaling involving the ephrins and Eph receptors during brain development.

Because the protein-tyrosine phosphatase (PTP) Syp associates with the tyrosine-phosphorylated platelet-derived growth factor beta receptor (beta PDGFR), the beta PDGFR is a likely Syp substrate. We tested this hypothesis by determining whether recombinant Syp (rSyp) and a control PTP, recombinant PTP1B (rPTP1B), were able to dephosphorylate the beta PDGFR. The beta PDGFR was phosphorylated at multiple tyrosine residues in an in vitro kinase assay and then incubated with increasing concentrations of rSyp or rPTP1B. While the receptor was nearly completely dephosphorylated by high concentrations of rPTP1B, receptor dephosphorylation by rSyp plateaued at approximately 50%. Two-dimensional phosphopeptide maps of the beta PDGFR demonstrated that rSyp displayed a clear preference for certain receptor phosphorylation sites; the most efficiently dephosphorylated sites were phosphotyrosines (Tyr(P)-771 and -751, followed by Tyr(P)740, while Tyr(P)-1021 and Tyr(P)-1009 were very poor substrates. In contrast, rPTP1B displayed no selectivity for the various rPTP1B displayed no selectivity for the various beta PDGFR tyrosine phosphorylation sites and dephosphorylated all of them with comparable efficiency. A Syp construct that lacked the SH2 domains was still able to discriminate between the various receptor phosphorylation sites, although less effectively than full-length Syp. These in vitro studies predicted that Syp can dephosphorylate the receptor in vivo. Indeed, we found that a beta PDGFR mutant (F1009) that associates poorly with Syp, had a much slower in vivo rate of receptor dephosphorylation than the wild type receptor. In addition, the GTPase-activating protein of Ras (GAP) and phosphatidylinositol 3-kinase were less stably associated with the wild type beta PDGFR than with the F1009 receptor. These findings are consistent with the in vitro experiments showign that Syp prefers to dephosphorylate sites on the beta PDGFR, that are important for binding phosphatidylinositol 3-kinase (Tyr(P)-740 and Tyr(P)-751) and GAP (Tyr(P)-771). These studies reveal that Syp is a substrate-selective PTP and that both the catalytic domain and the SH2 domains contribute to Syp's ability to choose substrates. Furthermore, it appears that Syp plays a role in PDGF-dependent intracellular signal relay by selectively dephosphorylating the beta PDGFR and thereby regulating the binding of a distinct group of receptor-associated signal relay enzymes.

Triple negative breast cancer (TNBC) is a highly heterogeneous disease, notoriously challenging to treat with standard chemotherapy options, and therefore is an area of intense focus for discovery of novel effective combination therapies. Here we used a previously described technology platform called CIVO, which enables assessment of multiple drugs and drug combinations simultaneously in living tumors, to identify drug combinations that result in synergistic anti-tumor activity in the HCC1187 model of TNBC. Our study focused on agents that combine with Voruciclib, a novel clinical stage oral CDK inhibitor with potent activity ( Citation Format: Joyoti Dey, Joseph Casalini, Sally Ditzler, Matt Biery, Angela Merrell, Derek Thirstrup, Marc Grenley, Richard Klinghoffer. Voruciclib, a clinical stage oral CDK inhibitor, sensitizes triple negative breast cancer xenografts to proteasome inhibition. [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 2835.

The multi-protein beta-catenin destruction complex tightly regulates beta-catenin protein levels by shuttling beta-catenin to the proteasome. Glycogen synthase kinase 3beta (GSK3beta), a key serine/threonine kinase in the destruction complex, is responsible for several phosphorylation events that mark beta-catenin for ubiquitination and subsequent degradation. Because modulation of both beta-catenin and GSK3beta activity may have important implications for treating disease, a complete understanding of the mechanisms that regulate the beta-catenin/GSK3beta interaction is warranted. We screened an arrayed lentivirus library expressing small hairpin RNAs (shRNAs) targeting 5,201 human druggable genes for silencing events that activate a beta-catenin pathway reporter (BAR) in synergy with 6-bromoindirubin-3'oxime (BIO), a specific inhibitor of GSK3beta. Top screen hits included shRNAs targeting dihydrofolate reductase (DHFR), the target of the anti-inflammatory compound methotrexate. Exposure of cells to BIO plus methotrexate resulted in potent synergistic activation of BAR activity, reduction of beta-catenin phosphorylation at GSK3-specific sites, and accumulation of nuclear beta-catenin. Furthermore, the observed synergy correlated with inhibitory phosphorylation of GSK3beta and was neutralized upon inhibition of phosphatidyl inositol 3-kinase (PI3K). Linking these observations to inflammation, we also observed synergistic inhibition of lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (TNFalpha, IL-6, and IL-12), and increased production of the anti-inflammatory cytokine IL-10 in peripheral blood mononuclear cells exposed to GSK3 inhibitors and methotrexate. Our data establish DHFR as a novel modulator of beta-catenin and GSK3 signaling and raise several implications for clinical use of combined methotrexate and GSK3 inhibitors as treatment for inflammatory disease.

We report the initial clinical findings of a device platform to test multiple therapeutic options simultaneously in individual living tumors within a clinical patient population, detailing for the first time comparative effects of different drugs within individual intact tumors. Most early cancer drug discovery is performed under in vitro conditions in cell-based models that poorly represent the disease they are intended to represent and have a poor track record for predicting success in subsequent clinical trials. To enable in vivo analysis of anti-cancer agent efficacy at earlier stages of drug development, and to potentially enable toxicity-sparing assessment of novel agents in the oncology clinic, we have developed a technology platform called CIVO™. CIVO allows for simultaneous assessment of up to eight drugs or drug combinations in a single solid tumor while the tumor is still in the patient. Controlled microinjection-based delivery of doxorubicin, docetaxel, mafosfamide, and gemcitabine was tested in twelve patients in the canine sarcoma clinic. Drugs were co-injected in a columnar array with UV fluorescent beads resulting in easy-to-identify bands of drug at 95% of the injection sites, each at a distinct position of the patient's tumor. Tumors were resected 72h following microinjection and were subjected to multiplexed analyses for tumor response which included drug-induced ablation of sarcoma cells, apoptosis, DNA damage, mitotic arrest, immune infiltration, and feedback activation of oncogenic pathways. The CIVO-introduced drug microdoses induced spatially-defined graded, and mechanism-specific cellular changes around sites of drug exposure in a drug and patient-specific manner. Consistent with the use of doxorubicin as first line therapy in the soft tissue sarcoma clinic, the frequency and extent of response of localized tumor kill induced by Doxorubicin (6/9 patients) exceeded those of all other agents tested, with Docetaxel being next most effective (2/6 patients), and Gemcitabine being the least effective (0/9 patients). Interestingly, almost all confirmed sites of response showed evidence of mTOR pathway upregulation, suggesting that mTOR pathway activation represents a potential common mechanism of chemo-resistance across sarcoma patients exists to multiple chemo-toxic agents. We are currently exploring combinations of 1st line sarcoma drugs with mTOR inhibitors in the canine sarcoma clinic with CIVO. This data represents the first time differential effects of multiple drugs have been captured within individual intact tumors in a clinical population. Along with early responses observed in the human clinic, this sets the stage for application of this technology to identify which novel agents are likely to succeed or fail in subsequent clinical trials. Citation Format: Alicia Moreno Gonzalez, Jason Frazier, William Kerwin, Jessica Bertout, Joseph Casalini, Sally Ditzler, Nathan Caffo, Richard A. Klinghoffer. A platform to test multiple therapies simultaneously in the intact tumors of cancer patients: Initial clinical experience. [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 2020. doi:10.1158/1538-7445.AM2015-2020

Guided by the belief that the most important setting for understanding tumor response to drugs is the human patient, we developed a technology called CIVO. CIVO enables analysis of up to 8 therapies simultaneously in a patient's tumor, without inducing systemic toxicity and while maintaining the integrity of the native tumor microenvironment.

e22177 Background: Cancer drug developers face a fundamental challenge in the way new candidate drugs are evaluated. Early stage compounds are tested in preclinical models that lack important compo...