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// Joseph Dosch 1, * , Elizabeth Ziemke 1, * , Shanshan Wan 2 , Kathryn Luker 1 , Theodore Welling 2 , Karin Hardiman 2 , Eric Fearon 3 , Suneetha Thomas 4 , Matthew Flynn 4 , Jonathan Rios-Doria 4 , Robert Hollingsworth 4 , Ronald Herbst 4 , Elaine Hurt 4, # and Judith Sebolt-Leopold 1, # 1 Department of Radiology, University of Michigan, Ann Arbor, MI 48109, USA 2 Department of Surgery, University of Michigan, Ann Arbor, MI 48109, USA 3 Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA 4 Department of Oncology Research, MedImmune, LLC, Gaithersburg, MD 20878, USA * These authors contributed equally to this work # Co-senior authors Correspondence to: Judith Sebolt-Leopold, email: jssl@med.umich.edu Keywords: ADAM17, TACE, colorectal, stem cell, Notch Received: November 24, 2016 Accepted: April 03, 2017 Published: May 10, 2017 ABSTRACT ADAM17 (a disintegrin and metalloproteinase 17)/TACE (TNFα converting enzyme) has emerged as a potential therapeutic target in colorectal cancer (CRC) and other cancers, due in part to its role in regulating various tumor cell surface proteins and growth factors and cytokines in the tumor microenvironment. The emergence of MEDI3622, a highly potent and specific antibody-based ADAM17 inhibitor, has allowed testing of the concept that targeting ADAM17 may be an important new therapeutic approach for CRC patients. We demonstrate that MEDI3622 is highly efficacious on tumor growth in multiple human CRC PDX models, resulting in improved survival of animals bearing tumor xenografts. MEDI3622 was further found to impact Notch pathway activity and tumor-initiating cells. The promising preclinical activity seen here supports further clinical investigation of this treatment approach to improve therapeutic outcome for patients diagnosed with metastatic CRC, including patients with KRAS-mutant tumors for whom other therapeutic options are currently limited.

Patients with many types of malignancy commonly harbor quiescent disseminated tumor cells in bone marrow. These cells frequently resist chemotherapy and may persist for years before proliferating as recurrent metastases. To test for compounds that eliminate quiescent cancer cells, we established a new 384-well 3D spheroid model in which small numbers of cancer cells reversibly arrest in G1/G0 phase of the cell cycle when cultured with bone marrow stromal cells. Using dual-color bioluminescence imaging to selectively quantify viability of cancer and stromal cells in the same spheroid, we identified single compounds and combination treatments that preferentially eliminated quiescent breast cancer cells but not stromal cells. A treatment combination effective against malignant cells in spheroids also eliminated breast cancer cells from bone marrow in a mouse xenograft model. This research establishes a novel screening platform for therapies that selectively target quiescent tumor cells, facilitating identification of new drugs to prevent recurrent cancer.

While several aberrant signaling pathways have been attributed to the formation and progression of pancreatic cancer, there is mounting evidence for the increased role of the Hedgehog (Hh) pathway in multiple aspects of pancreatic tumor development. The Hh pathway is a signaling cascade that plays an important role in cell patterning of multiple tissues and organs, including the development of the gastrointestinal system. While normal pancreatic tissue exhibits little Hh pathway activity, patients with pancreatic adenocarcinoma show high levels of Hh pathway signaling in both the tumor epithelia and the surrounding mesenchyme. Several recent studies have focused on this paracrine activation of Hh signaling in the tumor microenvironment and have provided evidence for how activation of this pathway may play roles in mediating cellular proliferation, metastasis, and resistance to therapy. Together, these findings present new insights into how modulation of this pathway may allow us to target multiple aspects of pancreatic tumor biology.

Tumor-initiating cells, also designated as cancer stem cells, are proposed to constitute a subpopulation of malignant cells central to tumorigenesis, metastasis, and treatment resistance. We analyzed the activity of the proteasome, the primary organelle for targeted protein degradation, as a marker of tumor- and metastasis-initiating cells. Using human and mouse breast cancer cells expressing a validated fluorescent reporter, we found a small subpopulation of cells with low proteasome activity that divided asymmetrically to produce daughter cells with low or high proteasome activity. Breast cancer cells with low proteasome activity had greater local tumor formation and metastasis in immunocompromised and immunocompetent mice. To allow flexible labeling of cells, we also developed a new proteasome substrate based on HaloTag technology. Patient-derived glioblastoma cells with low proteasome activity measured by the HaloTag reporter show key phenotypes associated with tumor-initiating cells, including expression of a stem cell transcription factor, reconstitution of the original starting population, and enhanced neurosphere formation. We also show that patient-derived glioblastoma cells with low proteasome activity have higher frequency of tumor formation in mouse xenografts. These studies support proteasome function as a tool to investigate tumor- and metastasis-initiating cancer cells and a potential biomarker for outcomes in patients with several different cancers.

Transforming growth factor-β (TGF-β) signaling regulates cell proliferation and differentiation, which contributes to development and disease. Upon binding TGF-β, the type I receptor (TGFBRI) binds TGFBRII, leading to the activation of the transcription factors SMAD2 and SMAD3. Using an RNA interference screen of the human kinome and a live-cell reporter for TGFBR activity, we identified the kinase BUB1 (budding uninhibited by benzimidazoles-1) as a key mediator of TGF-β signaling. BUB1 interacted with TGFBRI in the presence of TGF-β and promoted the heterodimerization of TGFBRI and TGFBRII. Additionally, BUB1 interacted with TGFBRII, suggesting the formation of a ternary complex. Knocking down BUB1 prevented the recruitment of SMAD3 to the receptor complex, the phosphorylation of SMAD2 and SMAD3 and their interaction with SMAD4, SMAD-dependent transcription, and TGF-β-mediated changes in cellular phenotype including epithelial-mesenchymal transition (EMT), migration, and invasion. Knockdown of BUB1 also impaired noncanonical TGF-β signaling mediated by the kinases AKT and p38 MAPK (mitogen-activated protein kinase). The ability of BUB1 to promote TGF-β signaling depended on the kinase activity of BUB1. A small-molecule inhibitor of the kinase activity of BUB1 (2OH-BNPP1) and a kinase-deficient mutant of BUB1 suppressed TGF-β signaling and formation of the ternary complex in various normal and cancer cell lines. 2OH-BNPP1 administration to mice bearing lung carcinoma xenografts reduced the amount of phosphorylated SMAD2 in tumor tissue. These findings indicated that BUB1 functions as a kinase in the TGF-β pathway in a role beyond its established function in cell cycle regulation and chromosome cohesion.

Survival rates associated with pancreatic cancer remain dismal despite advancements in detection and experimental treatment strategies. Genetically engineered mouse models of pancreatic tumorigenesis have gained considerable attention based on their ability to recapitulate key clinical features of human disease including chemotherapeutic resistance and fibrosis. However, it is unclear if transgenic systems exemplified by the KrasG12D/Trp53R172H/Pdx-1-Cre (KPC) mouse model recapitulate the functional heterogeneity of human pancreatic tumors harboring distinct cells with tumorigenic properties. To facilitate tracking of heterogeneous tumor cell populations, we incorporated a luciferase-based tag into the genetic background of the KPC mouse model. We isolated pancreatic cancer cells from multiple independent tumor lines and found that roughly 1 out of 87 cells exhibited tumorigenic capability. Notably, this frequency is significantly higher than reported for human pancreatic adenocarcinomas. Cancer stem cell (CSC) markers, including CD133, CD24, Sca-1, and functional Aldefluor activity, were unable to discriminate tumorigenic from nontumorigenic cells in syngeneic transplants. Furthermore, three-dimensional spheroid cultures originating from KPC tumors did not enrich for cells with stem-like characteristics and were not significantly more tumorigenic than cells cultured as monolayers. Additionally, we did not observe significant differences in response to gemcitabine or salinomycin in several isolated subpopulations. Taken together, these studies show that the hierarchical organization of CSCs in human disease is not recapitulated in a commonly used mouse model of pancreatic cancer and therefore provide a new view of the phenotypic and functional heterogeneity of tumor cells. Cancer Res; 75(21); 4582–92. ©2015 AACR.

Background & Aims Growth of many different tumor types requires a population of self-renewing cancer stem cells (CSCs). c-Met is a marker of normal mouse pancreatic stem and progenitor cells; we investigated whether it is also a marker of human pancreatic CSCs that might be developed as a therapeutic target. Methods We studied growth of primary human pancreatic adenocarcinoma in NOD SCID mice. The self-renewal capability of pancreatic cancer cells that expressed high levels of c-Met (c-Met high ) was assessed using in vitro sphere assays and compared with those that were c-Met negative or expressed low levels of c-Met. The tumorigenicity of c-Met high pancreatic cancer cells was evaluated in NOD SCID mice. Results c-Met high cells readily formed spheres, whereas c-Met–negative cells did not. Use of the c-Met inhibitor XL184 or c-Met knockdown with small hairpin RNAs significantly inhibited tumor sphere formation. c-Met high cells had increased tumorigenic potential in mice; those that expressed c-Met and CD44 (0.5%–5% of the pancreatic cancer cells) had the capability for self-renewal and the highest tumorigenic potential of all cell populations studied. In pancreatic tumors established in NOD SCID mice, c-Met inhibitors slowed tumor growth and reduced the population of CSCs when given alone or in combination with gemcitabine. Administration of XL184 for 2 weeks after cardiac injection of cancer cells prevented the development of metastases. Conclusions c-Met is a new marker for pancreatic CSCs. It is required for growth and metastasis of pancreatic tumors in mice and is a therapeutic target for pancreatic cancer.

Pancreatic cancer is a devastating disease that usually presents at a late stage that is not amenable to curative treatments. It is notoriously resistant to chemotherapy and radiation, and it is clear that new treatment strategies are needed to positively impact the dismal outcomes associated with this disease. While the initial dogma of tumor biology viewed all tumor cells as equal, recent findings in the field have lead to the discovery of heterogeneous populations of tumor cells, first in blood-borne derived cancers and now within multiple solid tumor organ systems that display a differential ability to repopulate the original tumor. Conceptualized from this finding was the stem-cell hypothesis for cancer, which suggests that only a specific subset of cancer cells within each tumor is responsible for tumor initiation and propagation, termed tumor initiating cells or cancer stem cells (CSCs). By using specific cell surface marker analysis, we have identified distinct populations of tumor cells from primary pancreatic adenocarcinomas that express the surface markers CD44, CD24, and ESA, which are responsible for the initiation of new tumor growth and exhibit characteristics that support the CSC hypothesis. The identification of these cells has opened up an exciting new paradigm on how we might view and devise new patient treatments for this devastating disease. Current investigations of these pancreatic CSCs have identified such signaling pathways as the Hedgehog and BMI-1 signaling pathways which are aberrantly regulated in this select population of cells. Further study of these CSCs and the important signaling pathways that control their function will provide us with powerful information on targeting new therapies to improve patient outcomes.

Cellular heterogeneity in cancer was observed decades ago by studies in mice which showed that distinct subpopulations of cells within a tumor mass are capable of driving tumorigenesis. Conceptualized from this finding was the stem-cell hypothesis for cancer, which suggests that only a specific subset of cancer cells within each tumor is responsible for tumor initiation and propagation, termed tumor initiating cells or cancer stem cells (CSCs). Recent data has been provided to support the existence of CSCs in human blood cell–derived cancers and solid organ tumors of the breast, brain, prostate, colon, and skin. Study of human pancreatic cancers has also revealed a specific subpopulation of cancer cells that possess the characteristics of CSCs. These pancreatic cancer stem cells express the cell surface markers CD44, CD24, and epithelial-specific antigen, and represent 0.5% to 1.0% of all pancreatic cancer cells. Along with the properties of self-renewal and multilineage differentiation, pancreatic CSCs display upregulation of important developmental genes that maintain self-renewal in normal stem cells, including Sonic hedgehog (SHH) and BMI-1. Signaling cascades that are integral in tumor metastasis are also upregulated in the pancreatic CSC. Understanding the biologic behavior and the molecular pathways that regulate growth, survival, and metastasis of pancreatic CSCs will help to identify novel therapeutic approaches to treat this dismal disease.

There continue to be many questions about the frequency and phenotype of tumor-initiating cells or cancer stem cells (CSCs) which may play an important role in cancer metastasis and drug resistance in several tumor types, including pancreatic cancer. The KrasG12D, Trp53R172H, and Pdx-1-Cre (KPC) induced model of pancreatic cancer has gained considerable attention for its ability to replicate the clinical features of the disease. Here, we report the generation of a novel syngeneic xenograft model of luciferase-tagged pancreatic tumor cells (KPCL) to facilitate the investigation of the tumorigenic capacity of this cancer model. Limiting dilution analysis of bulk KPCL tumor cells revealed that 1/87 cells were capable of forming new tumors, a number considerably higher than reported for human pancreatic adenocarcinomas. Cell surface markers CD44, CD24, CD133, c-Met, and functional ALDEFLUOR activity, previously shown to mark CSC populations in human and mouse pancreatic tumors, were heterogeneous across a large panel of spontaneous KPCL tumors. Additional testing of CD133 and ALDEFLUOR populations were unable to enrich for tumorigenic populations when transplanted into syngeneic mice. Additionally, spheroid cultures initiated from KPCL tumors did not enrich for cells with stem-like characteristics and were not significantly more tumorigenic than KPCL cells cultured as monolayers. These studies demonstrate that the tumor-forming potential of KPC cells is common, and may not fit the hierarchically organized CSC model that is reported for the human disease. Citation Format: Joseph S. Dosch, Elizabeth K. Ziemke, Amrith Shettigar, Alnawaz Rehemtulla, Judith S. Sebolt-Leopold. Evaluation of tumor-initiating cell populations in the KPC mouse model of pancreatic cancer. [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 1400. doi:10.1158/1538-7445.AM2015-1400

ADAM17/TACE (TNFα converting enzyme) has been implicated to play a role in the drug resistance of colorectal cancers. The present study addresses the hypothesis that MEDI3622, a specific ADAM17 monoclonal antibody known to possess both human and mouse cross reactivity, represents a viable therapeutic strategy to improve outcome in the treatment of metastatic colorectal cancer (mCRC). We have generated both in vitro and in vivo data in patient-derived CRC models to provide compelling support for further exploration of this treatment strategy. A heterogeneous panel of low passage colorectal patient-derived xenografts (PDX) has been established from biospecimens procured during patient surgeries conducted at University of Michigan. All xenograft models have been genomically profiled and their histologies mimicked those observed in the original samples. As anticipated, approximately half of these models harbor an oncogenic KRAS mutation and exhibit widely differing histologies, consistent with the heterogeneity encountered in the clinic. Efficacy testing of MEDI3622 has shown that this agent significantly impaired tumor growth in 3 of the 5 PDX models examined, including one model that is KRASMT. Furthermore, cancer stem cell function in these CRC models is negatively impacted by MEDI3622, reducing tumor initiation frequencies in tumor re-implantation studies at the conclusion of treatment. Therefore our results suggest that a monoclonal antibody approach that targets ADAM17 may provide an attractive therapeutic strategy for the treatment of metastatic colorectal cancer both by reducing tumor growth and inhibiting cancer stem cells. Citation Format: Joseph Dosch, Elizabeth Ziemke, Theodore Welling, Karin Hardiman, Juidth Sebolt-Leopold, Emil Michelotti, Robert Hollingsworth, Elaine Hurt. Monoclonal antibody targeting of ADAM17 is an effective treatment for metastatic colorectal cancer resulting in tumor growth control and reductions of cancer stem cells. [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 975. doi:10.1158/1538-7445.AM2015-975

The transforming growth factor-beta (TGF-β) family of cytokines regulates many processes such as immune suppression, angiogenesis, wound healing and epithelial to mesenchymal transition (EMT. Early in tumorigenesis, when epithelial cells retain exquisite growth sensitivity to this ligand, TGF-β signaling elicits a tumor suppressing activity. However, transformed cells become refractory to TGF-β-mediated growth inhibition and acquire a phenotype wherein the intracellular signaling circuitry of the cells is altered, leading to tumorigenic and metastatic effects in response to TGF-β exposure. Although TGF-β activating pathways have been studied, the molecular participants are poorly defined. Here we identify budding uninhibited by benzimidazoles-1 (Bub1) as an integral component of canonical and non-canonical TGF-β signaling pathways, where Bub1 is required for TGFBRI-TGFBRII complex formation and activation. Bub1-depleted cells exhibited reductions in TGF-β dependent Smad2/3 phosphorylation, recruitment of Smad2/3 to the TGFBRI-II complex, PI3K/Akt and p38MAPK activation, Smad binding element driven promoter activity (SBE4-Luc), and invasion and migration. Furthermore, a targeted small molecule inhibitor of Bub1 kinase activity (2OH-BNPP1), as well as an inactive kinase mutant of Bub1, abrogated ligand mediated TGF-β signaling and phenotypic response. These studies demonstrate a role for the Bub1 kinase in mediating TGF-β dependent signaling beyond its established function in cell-cycle regulation and chromosome cohesion and uncover the underlying basis for the pleiotropic cellular response commonly observed upon activation of the pathway. Citation Format: Shyam Nyati, Katrina Schinske-Sebolt, Sethuramasundaram Pitchiaya, Katerina Chekhovskiy, Areeb Chator, Nauman Chaudhry, Joseph Dosch, Marcian E. Van Dort, Varambally, Kumar-Sinha, Nyati, Ray, Walter, Sooryanarayana Varambally, Chandan Kumar-Sinha, Mukesh K. Nyati, Dipankar Ray, Nils G. Walter, Hongtao Yu, Brian D. Ross, Alnawaz Rehemtulla. Bub1 is a key regulator of TGF-β signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1137. doi:10.1158/1538-7445.AM2014-1137

Aberrant hyperactivation of KRAS plays a prominent role in tumor initiation and progression of a broad spectrum of human malignancies. The incidence of KRAS mutations is especially high in colorectal cancers, where it occurs at a frequency of roughly 50%. The present study addresses the hypothesis that dual targeting of CDK4 and MEK represents a viable treatment strategy for the subpopulation of colorectal cancers exhibiting hyperactivated KRAS signaling. We have generated both in vitro and in vivo data in highly passaged colorectal xenograft models to provide compelling support for further exploration of this combination treatment strategy. We have now turned to the study of primary human models that are anticipated to be more predictive of ultimate clinical activity in patients. A heterogeneous panel of low passage colorectal patient-derived xenografts (PDX) has been established from biospecimens procured during patient surgeries conducted at our institution (n=12). All xenograft models have been genomically profiled and their histologies mimicked those observed in the original samples. As anticipated, approximately half of these models harbor an oncogenic KRAS mutation and exhibit widely differing histologies, consistent with the heterogeneity encountered in the clinic. Immunoblotting analysis has further revealed that that the majority (>80%) are positive for both phosphorylated RB and constitutive pERK expression and are therefore candidates for treatment regimens combining inhibitors of CDK4 and MEK. Employing the CDK4 inhibitor palbociclib and multiple MEK inhibitors, in vivo efficacy studies are ongoing against our colorectal PDX panel. Preliminary data have shown a higher frequency of regressions in the combination arm compared to either single agent arm at the respective maximum tolerated doses. Our results therefore suggest that combined inhibition of CDK4 and MEK may provide an attractive therapeutic strategy for the treatment of colorectal cancer. Citation Format: Elizabeth Ziemke, Joseph Dosch, Amrith Shettigar, Shanshan Wan, Theodore Welling, Karin Hardiman, Judith Sebolt-Leopold. Dual targeting of CDK4 and MEK as a combination treatment strategy for metastatic colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4541. doi:10.1158/1538-7445.AM2014-4541

Pancreatic adenocarcinoma is a highly aggressive disease which is usually diagnosed in an advanced state and for which there are little/no effective therapies. The growth of many cancers depends on a subpopulation of self-renewing cells, termed cancer stem cells (CSC). We have previously identified such a CSC population in human pancreatic cancers which possess the cell surface marker phenotype CD44+CD24+ESA+. C-Met, a member of the receptor tyrosine kinases (RTKs) family and receptor for the hepatocyte growth factor (HGF) ligand, has been recently described as a marker of normal mouse pancreatic stem/progenitor cells. We hypothesized that c-Met may function as a cell surface marker and therapeutic target for human pancreatic CSCs. Studies were performed in low passage primary human pancreatic adenocarcinoma xenografts (n = 3) established in NOD-SCID mice. Self-renewal capacity of c-Methigh pancreatic cancer cells was assessed using in vitro sphere assays and in vivo tumorigenicity of c-Methigh pancreatic cancer cells was evaluated by cell implantation in NOD/SCID mice. In vitro studies demonstrated that c-Methigh cells formed spheres in non-adherent cultures, while c-Met− cells did not. Use of the c-Met inhibitor XL184 or c-Met knockdown with shRNA significantly inhibited tumor sphere formation. Pancreatic cancer cells expressing high c-Met had increased tumorigenic potential in vivo and cells expressing both CD44 and c-Methigh, representing 0.5-5% of pancreatic cancer cells, demonstrated the highest tumorigenic potential of all populations studied to date. C-Methigh CD44+ expressing pancreatic cancer cells demonstrated the capacity for self-renewal and production of differentiated progeny, properties that define CSCs. Further, c-Met inhibition of established primary human pancreatic tumors in NOD/SCID mice significantly inhibited tumor growth and depleted the pancreatic CSC population, when used alone or in combination with gemcitabine. Inhibition of c-Met also prevented the developmental of metastases using an intracardiac injection model. In this report, we define c-Met as a new marker for pancreatic CSC and define c-MethighCD44+ cells as a highly tumorigenic pancreatic CSC population. Our data also indicate that targeting c-Met may be useful in eradicating a CSC population and inhibiting the development of metastases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-257.